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Cortical sinus probing, S1P1-dependent entry and flow-based capture of egressing T cells.

Grigorova IL, Schwab SR, Phan TG, Pham TH, Okada T, Cyster JG - Nat. Immunol. (2008)

Bottom Line: Here we visualized the branched organization of lymph node cortical sinuses and found that after entry, some T cells were retained, whereas others returned to the parenchyma.T cells deficient in sphingosine 1-phosphate receptor type 1 probed the sinus surface but failed to enter the sinuses.We propose a multistep model of lymph node egress in which cortical sinus probing is followed by entry dependent on sphingosine 1-phosphate receptor type 1, capture of cells in a sinus region with flow, and transport to medullary sinuses and the efferent lymph.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California 94143, USA.

ABSTRACT
The cellular dynamics of the egress of lymphocytes from lymph nodes are poorly defined. Here we visualized the branched organization of lymph node cortical sinuses and found that after entry, some T cells were retained, whereas others returned to the parenchyma. T cells deficient in sphingosine 1-phosphate receptor type 1 probed the sinus surface but failed to enter the sinuses. In some sinuses, T cells became rounded and moved unidirectionally. T cells traveled from cortical sinuses into macrophage-rich sinus areas. Many T cells flowed from medullary sinuses into the subcapsular space. We propose a multistep model of lymph node egress in which cortical sinus probing is followed by entry dependent on sphingosine 1-phosphate receptor type 1, capture of cells in a sinus region with flow, and transport to medullary sinuses and the efferent lymph.

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Related in: MedlinePlus

Directional flow and T cell retention inside LYVE-1+ cortical sinuses(a) Three-dimensional reconstruction of LYVE-1+ structures imaged by two-photon intravital microsopy of inguinal LN. The structure was subdivided into macrophage-rich sinus area (red) and cortical sinuses (yellow) as described in the Methods. Colored tracks represent trajectories of Edg1+/+ T cells inside various interconnected regions of LYVE-1+ positive sinuses. See also Supplementary movie 5. (b) Superimposed tracks of cells in regions I, II (first 2 panels) and IV, V (last 2 panels) of a, in the xy plane, setting the starting coordinates to the origin. Scale bar, 30 μm. (c) Axis ratio of cells outside LYVE-1+ cortical sinuses (white triangle), in the regions of the sinuses with random cell movement (gray diamond) and in the regions with directional cell movement (gray circle). Data are combined from two intravital experiments (2 mice) and medians are shown by horizontal lines. (d) Two-photon microscopy image of T cell morphology in the parenchyma and inside a LYVE-1+ cortical sinus with directional cell movement. The image is a 6 μm z projection and the cells and structures were labeled as indicated. (e) Velocities of Edg1+/+ T cells from experiment illustrated in a. Velocities are shown for cells outside LYVE-1+ cortical sinuses (white triangles), and in sinus regions without flow (diamonds) and with flow (circles). Color-coding corresponds to the cortical sinus regions in a. (f) Velocities of Edg1+/+ T cells from three intravital experiments (3 mice). Median velocities were calculated for each period of time that cells spent outside sinuses (white triangles) or inside sinuses without flow (diamonds) and with flow (circles). (g) Distribution of time spent by Edg1+/+ T cells inside cortical LYVE-1+ sinuses in regions without flow (top panel) and with flow (lower panel). Data are pooled from three intravital experiments (3 mice). Light gray bars indicate tracks that entered and left the sinus within the tracking time. Dark gray bars indicate cells that transmigrated inside before the tracking began or were still inside at the end of the tracking. (h) Fraction of cells inside LYVE-1+ cortical sinuses that returned to the parenchyma within 30 min of tracking in regions without flow and with flow. Data are from three intravital experiments (3 mice). Bars indicate means. (i) Trajectories of two T cells tracked in green and blue migrating within a LYVE-1+ cortical sinus from a region without flow into an adjacent region where they are “captured” by flow at the position indicated by the red arrow. Image shows a fragment of region II from a. (j and k) Changes in the apparent velocities and axis ratio of the T cells shown in i. Red arrow indicates the location where the green and blue tracks cross into the region of flow. Velocity data are shown for the entire track duration whereas cell shape index data are shown for part of each track as indicated, where time zero indicates the start of imaging. Dashed lines indicate the median for cells in the absence of flow (long dashes) and in the presence of flow (short dashes), from c and f. Data in a, b, e are from one experiment that is representative of three (3 mice) and i-k are from one experiment that is representative of two (2 mice).
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Figure 4: Directional flow and T cell retention inside LYVE-1+ cortical sinuses(a) Three-dimensional reconstruction of LYVE-1+ structures imaged by two-photon intravital microsopy of inguinal LN. The structure was subdivided into macrophage-rich sinus area (red) and cortical sinuses (yellow) as described in the Methods. Colored tracks represent trajectories of Edg1+/+ T cells inside various interconnected regions of LYVE-1+ positive sinuses. See also Supplementary movie 5. (b) Superimposed tracks of cells in regions I, II (first 2 panels) and IV, V (last 2 panels) of a, in the xy plane, setting the starting coordinates to the origin. Scale bar, 30 μm. (c) Axis ratio of cells outside LYVE-1+ cortical sinuses (white triangle), in the regions of the sinuses with random cell movement (gray diamond) and in the regions with directional cell movement (gray circle). Data are combined from two intravital experiments (2 mice) and medians are shown by horizontal lines. (d) Two-photon microscopy image of T cell morphology in the parenchyma and inside a LYVE-1+ cortical sinus with directional cell movement. The image is a 6 μm z projection and the cells and structures were labeled as indicated. (e) Velocities of Edg1+/+ T cells from experiment illustrated in a. Velocities are shown for cells outside LYVE-1+ cortical sinuses (white triangles), and in sinus regions without flow (diamonds) and with flow (circles). Color-coding corresponds to the cortical sinus regions in a. (f) Velocities of Edg1+/+ T cells from three intravital experiments (3 mice). Median velocities were calculated for each period of time that cells spent outside sinuses (white triangles) or inside sinuses without flow (diamonds) and with flow (circles). (g) Distribution of time spent by Edg1+/+ T cells inside cortical LYVE-1+ sinuses in regions without flow (top panel) and with flow (lower panel). Data are pooled from three intravital experiments (3 mice). Light gray bars indicate tracks that entered and left the sinus within the tracking time. Dark gray bars indicate cells that transmigrated inside before the tracking began or were still inside at the end of the tracking. (h) Fraction of cells inside LYVE-1+ cortical sinuses that returned to the parenchyma within 30 min of tracking in regions without flow and with flow. Data are from three intravital experiments (3 mice). Bars indicate means. (i) Trajectories of two T cells tracked in green and blue migrating within a LYVE-1+ cortical sinus from a region without flow into an adjacent region where they are “captured” by flow at the position indicated by the red arrow. Image shows a fragment of region II from a. (j and k) Changes in the apparent velocities and axis ratio of the T cells shown in i. Red arrow indicates the location where the green and blue tracks cross into the region of flow. Velocity data are shown for the entire track duration whereas cell shape index data are shown for part of each track as indicated, where time zero indicates the start of imaging. Dashed lines indicate the median for cells in the absence of flow (long dashes) and in the presence of flow (short dashes), from c and f. Data in a, b, e are from one experiment that is representative of three (3 mice) and i-k are from one experiment that is representative of two (2 mice).

Mentions: We next asked whether we could detect directional cell movement within cortical sinuses. In the course of multiple imaging experiments we observed stable lymph flow through the subcapsular sinus in the first ∼2 h after anesthesia and surgery, and this was confirmed by the efficient drainage of subcutaneously injected phycoerythrin (PE) into medullary sinuses (Supplementary Movie 4 online). However, at later times the flow was reduced, in agreement with the requirement for active muscular activity to maintain maximal peripheral lymph flow16, 17. We therefore focused our imaging experiments testing for cortical sinus flow during the first 2 h after anesthesia and surgery. To facilitate tracking of cells within cortical sinuses we used Imaris software (see Methods) to generate a reconstruction of the sinus structures, distinguishing the cortical sinuses from macrophage-rich sinuses near the capsule (Fig. 4a and Supplementary Movie 5 online). Reconstructions were generated by tracing the outline of the sinus in each individual z-plane at several time points (Supplementary Fig. 2b-d online). By examining cell behavior within the lumen of several cortical sinus branches it was evident that in some, usually larger, branches the majority of T cells moved in the same direction with almost uniform intralumenal paths (Fig. 4a and Supplementary Movie 5online). When the tracks for a given branch were plotted on x–y plots the unidirectional flux of T cells in these branches was striking (Fig. 4b, branches IV and V in Fig. 4a) as opposed to the adjacent branches that exhibited essentially random T cell migration (Fig. 4b, branches I and II in Fig. 4a). Rare cells were detected that moved along the wall of the sinus in a direction against the flow (Fig. 4b, brown track in the last panel). Comparison of cell morphologies suggested the cells in a sinus region with flow were more rounded than cells in other regions (Fig. 4c,d). Measurement of the longest and shortest cell dimensions showed that cells in a region of flow had an axis ratio close to one, consistent with a rounded morphology whereas cells in other regions often had an elongated morphology with a length that was two or three-fold greater than their width (Fig. 4c,d). It is notable that T cells in a region of flow shared rather uniform velocities (Fig. 4e). Although moving unidirectionally and apparently flowing rather than actively migrating, T cells in a region of flow showed slower overall velocities than cells in the T zone or in sinus regions without flow (Fig. 4e,f). Interestingly, cell velocities in the parenchyma and inside the sinuses without flow were not significantly different (Fig. 4e,f). Cells migrated for variable and often short amounts of time inside the cortical sinus areas without evidence of flow, and about 50% of the cells migrated back into the T zone within 30 min (Fig. 4g,h). By contrast, T cells in a sinus showing evidence of flow were strongly retained and very few cells left through the walls of the structure into the T zone (Fig. 4g,h). Occasionally cells that transited from a region of active migration to a region of flow were detected (Fig. 4i and Supplementary Fig. 3 online). Upon making this transition the cells underwent a reduction in velocity and became more rounded (Fig. 4j,k). Some T cells could also be seen migrating from the parenchyma into cortical sinuses in a region with flow and becoming captured in the flow (data not shown).


Cortical sinus probing, S1P1-dependent entry and flow-based capture of egressing T cells.

Grigorova IL, Schwab SR, Phan TG, Pham TH, Okada T, Cyster JG - Nat. Immunol. (2008)

Directional flow and T cell retention inside LYVE-1+ cortical sinuses(a) Three-dimensional reconstruction of LYVE-1+ structures imaged by two-photon intravital microsopy of inguinal LN. The structure was subdivided into macrophage-rich sinus area (red) and cortical sinuses (yellow) as described in the Methods. Colored tracks represent trajectories of Edg1+/+ T cells inside various interconnected regions of LYVE-1+ positive sinuses. See also Supplementary movie 5. (b) Superimposed tracks of cells in regions I, II (first 2 panels) and IV, V (last 2 panels) of a, in the xy plane, setting the starting coordinates to the origin. Scale bar, 30 μm. (c) Axis ratio of cells outside LYVE-1+ cortical sinuses (white triangle), in the regions of the sinuses with random cell movement (gray diamond) and in the regions with directional cell movement (gray circle). Data are combined from two intravital experiments (2 mice) and medians are shown by horizontal lines. (d) Two-photon microscopy image of T cell morphology in the parenchyma and inside a LYVE-1+ cortical sinus with directional cell movement. The image is a 6 μm z projection and the cells and structures were labeled as indicated. (e) Velocities of Edg1+/+ T cells from experiment illustrated in a. Velocities are shown for cells outside LYVE-1+ cortical sinuses (white triangles), and in sinus regions without flow (diamonds) and with flow (circles). Color-coding corresponds to the cortical sinus regions in a. (f) Velocities of Edg1+/+ T cells from three intravital experiments (3 mice). Median velocities were calculated for each period of time that cells spent outside sinuses (white triangles) or inside sinuses without flow (diamonds) and with flow (circles). (g) Distribution of time spent by Edg1+/+ T cells inside cortical LYVE-1+ sinuses in regions without flow (top panel) and with flow (lower panel). Data are pooled from three intravital experiments (3 mice). Light gray bars indicate tracks that entered and left the sinus within the tracking time. Dark gray bars indicate cells that transmigrated inside before the tracking began or were still inside at the end of the tracking. (h) Fraction of cells inside LYVE-1+ cortical sinuses that returned to the parenchyma within 30 min of tracking in regions without flow and with flow. Data are from three intravital experiments (3 mice). Bars indicate means. (i) Trajectories of two T cells tracked in green and blue migrating within a LYVE-1+ cortical sinus from a region without flow into an adjacent region where they are “captured” by flow at the position indicated by the red arrow. Image shows a fragment of region II from a. (j and k) Changes in the apparent velocities and axis ratio of the T cells shown in i. Red arrow indicates the location where the green and blue tracks cross into the region of flow. Velocity data are shown for the entire track duration whereas cell shape index data are shown for part of each track as indicated, where time zero indicates the start of imaging. Dashed lines indicate the median for cells in the absence of flow (long dashes) and in the presence of flow (short dashes), from c and f. Data in a, b, e are from one experiment that is representative of three (3 mice) and i-k are from one experiment that is representative of two (2 mice).
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Figure 4: Directional flow and T cell retention inside LYVE-1+ cortical sinuses(a) Three-dimensional reconstruction of LYVE-1+ structures imaged by two-photon intravital microsopy of inguinal LN. The structure was subdivided into macrophage-rich sinus area (red) and cortical sinuses (yellow) as described in the Methods. Colored tracks represent trajectories of Edg1+/+ T cells inside various interconnected regions of LYVE-1+ positive sinuses. See also Supplementary movie 5. (b) Superimposed tracks of cells in regions I, II (first 2 panels) and IV, V (last 2 panels) of a, in the xy plane, setting the starting coordinates to the origin. Scale bar, 30 μm. (c) Axis ratio of cells outside LYVE-1+ cortical sinuses (white triangle), in the regions of the sinuses with random cell movement (gray diamond) and in the regions with directional cell movement (gray circle). Data are combined from two intravital experiments (2 mice) and medians are shown by horizontal lines. (d) Two-photon microscopy image of T cell morphology in the parenchyma and inside a LYVE-1+ cortical sinus with directional cell movement. The image is a 6 μm z projection and the cells and structures were labeled as indicated. (e) Velocities of Edg1+/+ T cells from experiment illustrated in a. Velocities are shown for cells outside LYVE-1+ cortical sinuses (white triangles), and in sinus regions without flow (diamonds) and with flow (circles). Color-coding corresponds to the cortical sinus regions in a. (f) Velocities of Edg1+/+ T cells from three intravital experiments (3 mice). Median velocities were calculated for each period of time that cells spent outside sinuses (white triangles) or inside sinuses without flow (diamonds) and with flow (circles). (g) Distribution of time spent by Edg1+/+ T cells inside cortical LYVE-1+ sinuses in regions without flow (top panel) and with flow (lower panel). Data are pooled from three intravital experiments (3 mice). Light gray bars indicate tracks that entered and left the sinus within the tracking time. Dark gray bars indicate cells that transmigrated inside before the tracking began or were still inside at the end of the tracking. (h) Fraction of cells inside LYVE-1+ cortical sinuses that returned to the parenchyma within 30 min of tracking in regions without flow and with flow. Data are from three intravital experiments (3 mice). Bars indicate means. (i) Trajectories of two T cells tracked in green and blue migrating within a LYVE-1+ cortical sinus from a region without flow into an adjacent region where they are “captured” by flow at the position indicated by the red arrow. Image shows a fragment of region II from a. (j and k) Changes in the apparent velocities and axis ratio of the T cells shown in i. Red arrow indicates the location where the green and blue tracks cross into the region of flow. Velocity data are shown for the entire track duration whereas cell shape index data are shown for part of each track as indicated, where time zero indicates the start of imaging. Dashed lines indicate the median for cells in the absence of flow (long dashes) and in the presence of flow (short dashes), from c and f. Data in a, b, e are from one experiment that is representative of three (3 mice) and i-k are from one experiment that is representative of two (2 mice).
Mentions: We next asked whether we could detect directional cell movement within cortical sinuses. In the course of multiple imaging experiments we observed stable lymph flow through the subcapsular sinus in the first ∼2 h after anesthesia and surgery, and this was confirmed by the efficient drainage of subcutaneously injected phycoerythrin (PE) into medullary sinuses (Supplementary Movie 4 online). However, at later times the flow was reduced, in agreement with the requirement for active muscular activity to maintain maximal peripheral lymph flow16, 17. We therefore focused our imaging experiments testing for cortical sinus flow during the first 2 h after anesthesia and surgery. To facilitate tracking of cells within cortical sinuses we used Imaris software (see Methods) to generate a reconstruction of the sinus structures, distinguishing the cortical sinuses from macrophage-rich sinuses near the capsule (Fig. 4a and Supplementary Movie 5 online). Reconstructions were generated by tracing the outline of the sinus in each individual z-plane at several time points (Supplementary Fig. 2b-d online). By examining cell behavior within the lumen of several cortical sinus branches it was evident that in some, usually larger, branches the majority of T cells moved in the same direction with almost uniform intralumenal paths (Fig. 4a and Supplementary Movie 5online). When the tracks for a given branch were plotted on x–y plots the unidirectional flux of T cells in these branches was striking (Fig. 4b, branches IV and V in Fig. 4a) as opposed to the adjacent branches that exhibited essentially random T cell migration (Fig. 4b, branches I and II in Fig. 4a). Rare cells were detected that moved along the wall of the sinus in a direction against the flow (Fig. 4b, brown track in the last panel). Comparison of cell morphologies suggested the cells in a sinus region with flow were more rounded than cells in other regions (Fig. 4c,d). Measurement of the longest and shortest cell dimensions showed that cells in a region of flow had an axis ratio close to one, consistent with a rounded morphology whereas cells in other regions often had an elongated morphology with a length that was two or three-fold greater than their width (Fig. 4c,d). It is notable that T cells in a region of flow shared rather uniform velocities (Fig. 4e). Although moving unidirectionally and apparently flowing rather than actively migrating, T cells in a region of flow showed slower overall velocities than cells in the T zone or in sinus regions without flow (Fig. 4e,f). Interestingly, cell velocities in the parenchyma and inside the sinuses without flow were not significantly different (Fig. 4e,f). Cells migrated for variable and often short amounts of time inside the cortical sinus areas without evidence of flow, and about 50% of the cells migrated back into the T zone within 30 min (Fig. 4g,h). By contrast, T cells in a sinus showing evidence of flow were strongly retained and very few cells left through the walls of the structure into the T zone (Fig. 4g,h). Occasionally cells that transited from a region of active migration to a region of flow were detected (Fig. 4i and Supplementary Fig. 3 online). Upon making this transition the cells underwent a reduction in velocity and became more rounded (Fig. 4j,k). Some T cells could also be seen migrating from the parenchyma into cortical sinuses in a region with flow and becoming captured in the flow (data not shown).

Bottom Line: Here we visualized the branched organization of lymph node cortical sinuses and found that after entry, some T cells were retained, whereas others returned to the parenchyma.T cells deficient in sphingosine 1-phosphate receptor type 1 probed the sinus surface but failed to enter the sinuses.We propose a multistep model of lymph node egress in which cortical sinus probing is followed by entry dependent on sphingosine 1-phosphate receptor type 1, capture of cells in a sinus region with flow, and transport to medullary sinuses and the efferent lymph.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California 94143, USA.

ABSTRACT
The cellular dynamics of the egress of lymphocytes from lymph nodes are poorly defined. Here we visualized the branched organization of lymph node cortical sinuses and found that after entry, some T cells were retained, whereas others returned to the parenchyma. T cells deficient in sphingosine 1-phosphate receptor type 1 probed the sinus surface but failed to enter the sinuses. In some sinuses, T cells became rounded and moved unidirectionally. T cells traveled from cortical sinuses into macrophage-rich sinus areas. Many T cells flowed from medullary sinuses into the subcapsular space. We propose a multistep model of lymph node egress in which cortical sinus probing is followed by entry dependent on sphingosine 1-phosphate receptor type 1, capture of cells in a sinus region with flow, and transport to medullary sinuses and the efferent lymph.

Show MeSH
Related in: MedlinePlus