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Am J Physiol Heart Circ Physiol 284: H2213-H2226, 2003. First published February 13, 2003; doi:10.1152/ajpheart.00999.2002
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Vol. 284, Issue 6, H2213-H2226, June 2003

Intravital microscopy comparing T lymphocyte trafficking to the spleen and the mesenteric lymph node

Mitchell H. Grayson1, Richard S. Hotchkiss4,6, Irene E. Karl2, Michael J. Holtzman3,5, and David D. Chaplin7

Divisions of 1 Allergy and Immunology, 2 Metabolism, and 3 Pulmonary and Critical Care Medicine, 4 Department of Medicine, and Departments of 5 Cell Biology and 6 Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110; and 7 Department of Microbiology, University of Alabama, Birmingham, Alabama 35294


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lymphocyte rolling velocity is determined largely by interactions between leukocyte alpha 4-integrin (CD49d) and L-selectin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in mesenteric postcapillary venules and Peyer's patch high endothelial venules (HEVs). The role of these interactions in other tissue sites of lymphocyte emigration is not known. With the use of real-time intravital confocal microscopy, we found that rolling velocities of T lymphocytes in the murine mesenteric lymph node (MLN) HEV also depend on L-selectin and CD49d. However, in the murine spleen, rolling velocities of T lymphocytes are not influenced by the loss of L-selectin and CD49d. With the use of FITC-dextran and TIE2-GFP mice, we further defined the microvascular compartments of the spleen and showed that adherence of T cells is localized to regions in the white pulp that are not lined by endothelial cells and have shear rates similar to bone marrow sinusoids. These results establish that T cell trafficking to the spleen differs from trafficking to other secondary lymphoid organs and suggest that the mechanical properties of the blood-filtering role of the spleen are important in T cell accumulation in the organ.

cell adhesion molecule; lymphocyte rolling; hemodynamic parameters; microvascular vessels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE INITIAL ENCOUNTER between naïve T cells and antigen generally occurs in secondary lymphoid tissues (24). This encounter requires an interaction between antigen-bearing antigen-presenting cells and rare antigen-specific naïve T cells. The organized structure of the secondary lymphoid tissues is thought to facilitate these cellular interactions as well as the interactions between antigen-specific T and B cells that underlie T cell help in the humoral response (11). Identification of the molecular signals that establish and maintain the normal cellularity of these tissues is therefore important for understanding how initial T cell sensitization is regulated.

The mechanisms governing the recruitment of naïve lymphocytes into the peripheral lymph node (PLN) and Peyer's patches have been largely defined. In the PLN, adhesive cellular interactions at the high endothelial venule (HEV) control the initial movement of naïve lymphocytes out of the circulatory compartment into the organized lymphoid tissue (6, 40). Naïve lymphocytes initially tether and roll on peripheral node addressin (PNAd) expressed on the HEV of the PLN (18). This rolling phenomenon has been well characterized in the PLN with the rolling velocities determined using intravital microscopy (44, 49). The ligand for PNAd on naïve lymphocytes is L-selectin (CD62L) (3). Rolling lymphocytes then adhere firmly via activation of their surface-expressed alpha Lbeta 2-integrin (CD11a/CD18; LFA-1), which interacts with ICAM-1 on the HEV surface (49). Firmly adherent lymphocytes then migrate across the HEV and, in an integrin-dependent manner, enter the lymphoid compartment of the PLN before moving to the appropriate locations in the tissue (6). The rules governing cell entry into the Peyer's patches are generally similar, although the initial rolling and tethering depends more on the interaction of L-selectin with the counterligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (4, 22). Subsequent firm adhesion is also dependent on MAdCAM-1 via its interaction with alpha 4beta 7-integrin on the lymphocyte (5, 8).

Like Peyer's patches, the mesenteric lymph node (MLN) expresses MAdCAM-1 on its HEV (20). Whereas the cellularity of the MLN was unaffected in mice deficient in either L-selectin or beta 7-integrin, it has been shown that mice deficient in both L-selectin and beta 7-integrin had dramatically reduced MLN cellularity (48). Similarly, over a 90-min time period, both L-selectin and beta 7-integrins were shown to be critical for populating the MLN with cells from the circulation. However, the relative contributions of L-selectin and alpha 4beta 7-integrin to the rolling phase of lymphocyte accumulation in the MLN have not been described, and direct measurements of lymphocyte rolling velocities or hemodynamic parameters in this tissue have not been reported.

The spleen is also a secondary lymphoid organ that expresses MAdCAM-1. In this case, MAdCAM-1 is detected in the marginal sinus, on the sinus lining cells overlaying the lymphoid compartment (20). Because of the location of the marginal sinus and its expression of MAdCAM-1, it has been postulated that the marginal sinus is the spleen functional equivalent of the lymph node HEV. However, few data exist to define where in the spleen lymphocytes exit the bloodstream and enter the lymphoid parenchyma (26). In fact, analyses of mice carrying targeted mutations of known adhesion molecules have shown no major alterations in the morphology or cellularity of the spleen lymphoid compartment (3, 5, 9, 21-23, 27, 39, 43, 47). Specifically, no role for MAdCAM-1 has been defined (20).

Analyses of spleen cellularity at equilibrium are poorly suited to defining the mechanisms controlling influx of cells into the tissue given the significant proportion (~5%) of cardiac output the spleen receives each cycle (34). This large amount of blood flow could permit minor pathways to maintain the cellularity of this tissue even when physiologically dominant mechanisms are experimentally blocked. Furthermore, specific interactions of adhesion molecules with their ligands have been reported to occur only within specific wall shear stresses (WSS)/wall shear rates (WSR) (25, 30, 31, 45, 49). Until now, no study of these values in the spleen has been undertaken, so it has been unclear whether these types of adhesive events could occur.

In this study, we used a novel real-time intravital laser scanning confocal microscopic technique to characterize the WSR in the murine spleen and to identify the site where T lymphocytes roll and adhere within the spleen. These data were then compared with those from the MLN. Our results suggest that trafficking of lymphocytes into the spleen occurs in a manner altogether different from other secondary lymphoid organs. It is tempting to speculate that this difference is related to the importance of the spleen in monitoring for and responding to blood-borne infectious organisms.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. C57BL/6, mixed C57BL/6 × 129S1/SvImJ (B6/129), FVB/N-TgN(TIE2GFP)287Sato (TIE2-GFP), and B6, 129S-L-selectin-deficient (B6,129-Selltm1Hyn) mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were housed for at least 1 wk before experimentation. No experimental differences were observed between male and female mice (6-12 wk old), and both were used throughout the study. Mice were housed in specific pathogen-free conditions with food and water provided ad libitum. The Washington University Institutional Committee for the Humane Use of Laboratory Animals approved all experiments.

No differences in rolling velocities or cellular accumulation in the MLN or spleen were noted using adoptive transfer of either C57BL/6 or B6/129 T cells into C57BL/6 recipients (data not shown); in these experiments, all recipients were C57BL/6 mice unless otherwise noted.

Reagents and antibodies. Low-azide, no endotoxin (LA/NE) anti-CD49d (clone R1-2) and IgG1-kappa control (anti-TNP) were obtained from B-D Pharmingen (San Diego, CA), as were anti-CD16/32 (clone 2.4G2) and phycoerythrin-conjugated anti-B220, anti-Thy1.2, and anti-IgG (clone MOP-1C) isotype controls. Transfluospheres (0.04 µm diameter) that excite at 488 nm and emit at 560 nm were obtained from Molecular Probes (Eugene, OR). Pertussis toxin (PTX) and 150-kDa FITC-dextran were obtained from Sigma (St. Louis, MO). Fluorescent polymer beads (3-µm diameter; excitation at 468 nm and emission at 508 nm) were obtained from Duke Scientific (Palo Alto, CA).

Cell purification and labeling. T lymphocytes (>= 95% pure) were obtained from donor splenocytes by negative selection using DYNA beads (DYNAL; Oslo, Norway) as described previously (12). By trypan blue (Sigma) exclusion, cells were always >= 97% viable. Cells (5 × 107 cells/ml) were labeled for 15 min at 37°C in the dark with 5 µM Oregon green 488 carboxylic acid succinimidyl ester (Molecular Probes) as previously described (12). This dye, which has emission and excitation spectra similar to fluorescein, is cleaved by intracellular esterases and remains intracellular (17). After being labeled, the cells were washed twice with sterile PBS (Life Technologies; Rockville, MD) at 4°C before being resuspended in PBS for injection. Cells (107) were injected retroorbitally in a total volume of 100-150 µl. When antibodies were used, 107 cells were mixed with 1 µg of the appropriate antibody (control IgG or anti-CD49d), loaded into syringes, and kept on ice for 30 min before being warmed to 37°C and injected into the recipient mouse.

Analysis of rolling and adhesion by confocal microscopy. The microscope used was a Nikon RCM-8000 (Nikon; Tokyo, Japan) equipped with an argon laser (Coherent; Palo Alto, CA). This laser emits at 351 and 488 nm. We used a Nikon fluor ×20/0.9 numerical aperture water immersion objective. The image size was 355 × 266 µm with a display of 512 × 480 pixels and an optical slice of 8.7 µm with a working distance of 800 µm (33).

Intravital microscopy was performed as previously described (12). Briefly, the mouse was anesthetized using a subcutaneous injection of 87 mg/kg ketamine (Fort Dodge Animal Health; Fort Dodge, IA) and 13 mg/kg xylazine (Burns Veterinary Supplies; Memphis, TN). Sterile saline (200 µl) was injected subcutaneously to maintain hydration, and a heating pad/lamp was used to maintain the mouse body temperature at 37°C.

The MLN was identified through a midline incision, and the mouse was placed in a modified petri dish with a central coverslip as described previously (12).

Blood flow was evaluated by intravenous injection of transfluospheres (10 µl of transfluospheres diluted in 100 µl of saline). We (12) have previously shown that these transfluospheres remain within the vasculature and appear red when excited at 488 nm. Once the blood flow had been visualized, labeled T cells or beads were injected, and images were captured on videotape for the 5 min immediately after the injection. Cells (107) or beads (3.7 × 107) in 100 µl of saline were injected retroorbitally per mouse.

For examination of cell trafficking in the spleen, a small incision was made in the left flank, and the spleen was exposed. The mouse and spleen then were positioned on the coverslip as previously described (12). Immediately after blood flow was assessed with transfluospheres, labeled T cells or beads were injected and observed for the 5 min after the injection.

Images were captured on VHS videotape (JVC of America) and analyzed at a later date.

Determination of lymphocyte rolling velocity and adherence. Every cell that appeared in the MLN HEV or the lymphoid compartment of the spleen during the first 5 min after intravenous transfer was followed. The distance the cell moved before adhering or leaving the field of view was measured, and the number of video frames required for this movement was determined. The velocity was calculated based on the fact that video frames are 1/30th of a second apart. The MLN HEV and lymphoid compartments of the spleen were identified by earlier injection of transfluospheres. Assessment of bead velocity was made similarly; however, because of the large number of beads seen in the organs, only 1 min of video was analyzed (this was the minute from 2-3 min after the injection).

Cells or beads were considered to be adherent if they did not move for 20 s. The velocity reported for adherent cells or beads was that observed just before the frames in which they became adherent. Additionally, cells or beads were classified as interacting or noninteracting based on their behavior with respect to the lining of the MLN HEV or spleen white pulp channels (see below).

The mean blood flow velocity (Vblood), WSR, and WSS were calculated as previously described (44, 46, 49). For flow through the MLN HEV, we used noninteracting T cells to determine Vblood; however, in the spleen, because it was rare to see noninteracting cells, we used the velocity of the front edge of a FITC-dextran injection as the Vblood value. These data were obtained from visualizing a minimum of three vessels of each vessel type from three to four C57BL/6 mice.

Statistical analysis. Student's t-test was used to assess statistical significance between means. Significance was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The structural differences between the MLN and spleen are characterized in Fig. 1. The spleen is much larger and is composed of both a lymphoid (white pulp) and reticuloendothelial compartment (red pulp). Prominent structures in the MLN are the postcapillary HEV. HEV have been thoroughly characterized in lymph nodes and Peyer's patches, where they serve as sites for lymphocytes to enter the lymphoid compartment from the circulation. A homologous structure has never been definitively identified in the spleen. On a morphological basis, due to its MAdCAM-1 expression, the marginal sinus, which separates lymphoid and nonlymphoid compartments of the spleen, has been postulated to be the spleen HEV equivalent; however, there are scant functional data supporting this hypothesis (26).


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  		Fig. 1.   A schematic representation of the mesenteric lymph node (MLN) high endothelial venule (HEV) and the spleen. Note that the side of the marginal sinus facing the lymphoid compartment of the spleen has mucosal addressin cell adhesion molecule (MAdCAM)-1 expressed on its surface, as does the HEV in the MLN. Whereas it is known that cells enter the MLN through the HEV, it is less clear where they exit the bloodstream to enter the spleen.

The only known ligands for MAdCAM-1 are L-selectin and alpha 4beta 7-integrin. Several studies using static assays have shown the primary role of L-selectin-MAdCAM-1 interactions in lymphocyte trafficking to the MLN as well as the lesser importance of alpha 4beta 7-integrin-MAdCAM-1 binding (4, 48). Intravital studies of vessels within the mesentery or HEV within Peyer's patches (both of which express MAdCAM-1) have shown that rolling of leukocytes can occur on MAdCAM-1 in both an L-selectin- and alpha 4beta 7-integrin-dependent fashion (4, 19). Therefore, we undertook these investigations to further characterize the behavior of lymphocyte trafficking to the MLN and spleen to determine the WSR in the MLN and spleen and to compare the contributions of the two known MAdCAM-1-binding ligands to T lymphocyte homing in both organs.

Analysis of lymphocyte trafficking in the spleen and MLN by intravital confocal microscopy. Although many intravital microscopic studies have examined peripheral lymph nodes, exposed mesenteric venules, and Peyer's patches, none have directly quantified the velocities of cell trafficking within the MLN or spleen. With the use of our real-time laser scanning confocal microscopy system for intravital experiments, we visualized the externalized murine MLN and spleen and studied T lymphocyte trafficking.

A representative set of images obtained from a murine MLN is shown in Fig. 2A.1 As observed, individual Oregon green-labeled T cells can be detected as firmly adherent, tethering, or rolling within MLN HEV. To verify that these adhesive events were not due to a mechanical property of the MLN, we injected 3-µm-diameter beads as well. As seen in Fig. 2B, these beads failed to interact with MLN HEV and rapidly passed through the organ. We chose 3-µm-diameter beads because beads of a larger diameter (>4 µm) failed to pass through the pulmonary circulation (50). In addition, it has been reported that a 4-µm-diameter inert bead is similar to a 7-µm-diameter neutrophil in its ability to move through the microvasculature (1). Therefore, we reasoned that a 3-µm-diameter bead would be similar to the smaller lymphocyte's size and should easily pass through the MLN or spleen if no mechanical event hindered it.


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  		Fig. 2.   Representative images of T lymphocytes or fluorescent beads moving through a MLN HEV. A: Oregon green 488-labeled T lymphocytes (green) moving through the MLN HEV. Cells can be seen to tether, roll, and adhere using this system. The asterisk marks an adherent cell, and two rolling cells are identified by white arrows. B: 3-µm-diameter fluorescent beads (green) do not interact with the MLN HEV, as shown in these images. A noninteracting bead is indicated by the white arrow. In both A and B, blood flow is indicated by transfluospheres (red) and the HEV is outlined with pink dashed lines in the first and last frames. The time from the first image is listed in each panel. Note the difference in time scale between the T cells (0.5 s between each frame) and the beads (0.04 s between each frame). The white bar represents 100 µm, and the blue arrows in the final frames show the routes that cells or beads took. Data shown are representative of similar results from at least 6 mice per condition.

A similar approach was used for the spleen. In Fig. 3A, a representative set of images demonstrates the movement of T lymphocytes within the spleen. As with the MLN, transfluospheres were used to identify bulk blood flow. Previously, we and others have shown that the majority of blood flow to the spleen (>90%) passes from arterioles through the white pulp and into draining veins without entering the red pulp (12, 38). Because we wanted to study the rolling of T lymphocytes at a site that might regulate trafficking from the circulation into the lymphoid compartment of the spleen, only cells moving through areas identified by transfluospheres were utilized in our analysis. The vast majority of adoptively transferred fluorescently labeled cells were found to remain within this region.


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  		Fig. 3.   Representative images of T lymphocytes or fluorescent beads moving through the lymphoid compartment of the spleen. A-C: three different Oregon green 488-labeled T lymphocytes (green) moving through the same spleen white pulp. D: 3-µm-diameter fluorescent beads (green) moving through the spleen white pulp. In A-C, cells can be seen to tether, roll, and adhere. The time from the first frame is indicated in each image. Note that in B the cell moves quite rapidly compared with A and C. In D, as with the T cells, the fluorescent beads can be seen to roll and adhere within the white pulp parenchyma. Unlike in the MLN (Fig. 2), the time intervals between the frames in A-D are quite similar. In A-D, blood flow is indicated by transfluospheres (red), and the white pulp is outlined with pink dashed lines in the first and last frames. The asterisk in the first frame of A-D indicates the site at which the index cell or bead originally appeared. The rolling cell or bead is indicated in each of the subsequent frames with a white arrow. The double white arrow in D indicates a second rolling bead. Note that in A-C the cells entered the lymphoid compartment through a similar location in the center of the white pulp. The white bar represents 100 µm, and the blue arrows in the final frames show the routes that cells or beads took. Data shown are representative of similar results from at least 6 mice per condition.

As in the MLN, fluorescent beads were visualized in the spleen. These beads were noted to "roll" and firmly "adhere" in the white pulp, as shown in Fig. 3B. Note, however, that the beads appeared to travel faster before firmly adhering than did lymphocytes; however, like lymphocytes, they first appeared near the center of the white pulp and then moved toward the periphery.

PTX has been shown to prevent the accumulation of circulating lymphocytes within the lymphoid compartment of the spleen (7). We treated T lymphocytes in vitro with PTX (100 ng/ml) and examined their resulting migration behavior within the spleen. Adoptively transferred cells were examined within the first 5 min after their intravenous injection. During this time period, no PTX-treated cells were detected within the spleen (data not shown). Although not further studied, we believe that these cells are trapped initially elsewhere in the vasculature. Nonetheless, the combination of these data, the bead data, and the transfluosphere data provide evidence that the cells appearing early in the spleen are indeed moving through the lymphoid compartment.

During our initial studies using this system, we noticed a qualitative difference in the trafficking characteristics of cells moving through the MLN versus the spleen. As shown in Fig. 4, some T cells in the MLN moved in a discontinuous manner, likely a result of direct interactions with the HEV. Indeed, in the MLN, these movements were always observed before a cell firmly adhered to the HEV wall. In the spleen, however, cells and beads usually moved at a steady velocity indicative of rolling until they suddenly stopped. At times, these cells would resume movement and exit the field of view-usually at a speed similar to that at which they originally arrived. These restart events were uncommon. In most cases, once the cells stopped in the spleen, they did not significantly move from that location during the 5 min of video capture. In addition to these cells that moved slowly (rolling) through the spleen, there were occasional cells or beads that would move very rapidly through the field of view. These cells moved at a high rate of speed and appeared to not interact with the splenic parenchyma as they traversed it.


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  		Fig. 4.   Rolling characteristics of individual cells in the MLN and spleen. A-F: instantaneous velocity tracings of 3 representative T cells from the MLN of 2 mice (A-C: data from mouse 1; D-F: data from mouse 2). Note the periods of no movement interspersed with rolling episodes. Contrast this with the tracings of T lymphocytes in the spleen (G-L). Again, cells from 2 different mice (G-I: data from mouse 1; J-L: data from mouse 2) were examined. In most cases in the spleen, cells rolled until they adhered. Once they stopped moving, cells were more apt to remain adherent in the spleen than in the MLN. Velocities (in µm/s) were calculated for each frame, and each data point represents a time interval of 0.03 s. The first 3 cells seen in the MLN or spleen were analyzed for their first 1.5 s in the field of view using the ×20 objective.

To more clearly identify the vascular structures in which the lymphocytes were moving, we injected 150-kDa FITC-dextran into mice and observed the spleen. As can be seen in Fig. 5A, blood flow into the spleen comes from well-circumscribed vessels into terminal "buds" or arborizations, where the blood appears to ooze out into surrounding tissue. These terminal vessels had a diameter of 7.2 ± 0.4 µm, and the velocity of blood flow was 300 ± 45 µm/s based on FITC-dextran injection (Table 1). As discussed below, T lymphocyte rolling velocities were slower than this, whereas the 3-µm-diameter beads rolled at a slightly more rapid rate. Upon higher power examination of these regions, we were able to identify a honeycomb-like vascular structure (Fig. 5A,c). We believe that the cells adhered within these structures (see below).


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  		Fig. 5.   Vascular characteristics of the spleen. A: injection of 150-kDa FITC-dextran (green) identifies the vessels in the spleen. As can be seen in a, well-circumscribed vessels end in terminal "buds," as indicated by the white arrow. The asterisks identify "honeycomb" regions where the blood has oozed from terminal buds. Note also that different areas of the spleen fill with blood at different rates, presumably due to the pulsating vessels (see below and text). In b, a higher-power image of the terminal bud (marked by the white arrow) from a is shown. Similarly, c shows a higher-power image of the honeycomb region from a (indicated by asterisks in both images). A pulsating vessel can be seen at the double white arrow in d. Note that this vessel bifurcates into additional vessels and does not itself end in a terminal bud. B: blood flow into the spleen white pulp occurs through the terminal vessels. Transfluospheres were injected into TIE2-GFP mice before these images were obtained to identify the white pulp. In the first frame (Pre-FITC), the green TEK-expressing endothelium-lined terminal vessels penetrate and are surrounded by the red transfluospheres. A magnified region of the area in the blue dashed box is provided in second frame (Inset). Note the red transfluospheres (marked by the white arrows) appearing at the ends of the TEK-expressing vessels (marked by a double white arrow). Shortly after this image was taken, 150-kDa FITC-dextran was injected to show the route of blood flow. The numbers at the bottom left of the subsequent frames indicate the number of seconds after the FITC-dextran was injected. Note that blood enters the white pulp through a single projection (at the white arrow) and then moves into terminal arborizations that end in the honeycomb region (marked by a double white arrows). The blue dashed box in the first image after injection (0 s) represents the same region shown in the inset. The slight shift in the position of the field of view occurred during the injection of FITC-dextran. C: T cell and polymer beads adhere outside of endothelium-lined vessels. Green labeled T lymphocytes (a and b) or 3-µm-diameter polymer beads (c and d) were injected into TIE2-GFP mice, and the spleen was visualized. These representative images are of adherent cells or beads, and white arrows mark the TIE2-GFP-expressing endothelium. Note that none of the T cells or beads are located within the vessels that are TEK+ endothelium lined, although the bead in d has adhered just beyond the TEK+ endothelium-lined vessel. D: representative images of a T cell traveling through a "channel" in the spleen. The channel walls are outlined in these images with a blue dashed line, and a T cell (marked by a white arrow) can be seen moving up and through the channel at ~90 µm/s. Note that this Oregon green 488-labeled cell appears yellow, indicating that it is colocalizing with the blood flow as identified by the red transfluospheres. The time (in s) from the first image is indicated at the bottom left of the frames. The asterisk indicates the site at which the T cell appeared in the channel. Unless otherwise indicated, in A-D the white bar represents 100 µm. Data shown are representative of similar results from at least 3 mice per condition.


                              
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  		Table 1.   Comparison of hemodynamic properties between the MLN and spleen vessels

In addition, we observed well-circumscribed pulsating vessels that experienced varying flow rates, ranging from no flow up to a maximum of 1,330 µm/s (mean ± SE: 384 ± 65 µm/s) with a mean diameter of 7.1 ± 0.5 µm (mean ± SE; Fig. 5A,d and Table 1) upstream of the terminal vessels. These vascular pulsations did not correlate with the mouse's respirations or heart rate (data not shown).

A comparison of relative hemodynamic values was made between the spleen vessels and MLN HEV. As can be seen in Table 1, Vblood was significantly higher in the MLN than in the spleen. Interestingly, although Vblood was similar between the terminal and pulsating vessels, only the terminating vessels had WSS that was similar to the MLN HEV.

To determine which, if any, of these structures were typical vessels with an endothelial cell lining, we performed similar experiments using TIE2-GFP mice. These mice express green fluorescent protein (GFP) under the Tek (formerly known as Tie2) promoter and are the best marker for intravital identification of endothelial cells (32). As seen in Fig. 5B, the well-circumscribed vessels have an endothelial cell layer; however, the terminal buds and honeycomb region do not. Interestingly, there was an abrupt end to the TEK-expressing endothelial cells where the terminal vessels emptied into the bud and honeycomb region. This was consistent with the drop in Vblood and WSR seen as the blood entered these regions as well.

To verify that T lymphocytes were adhering in this honeycomb region, we injected Oregon green 488-labeled T lymphocytes purified from the TIE2-GFP spleen into a TIE2-GFP recipient and visualized the spleen. As seen in Fig. 5C,a and b, T cells adhered in the region of blood flow just beyond the GFP+ endothelium. Interestingly, the 3-µm-diameter beads tended to adhere closer to the ends of the GFP+ endothelium (Fig. 5C,c and d) but were still within the honeycomb region.

With the use of the microscope's ×2 and ×4 zoom features (total magnification ×400 and ×800, respectively), we were able to more clearly visualize the channels in which the T lymphocytes appeared to adhere in the spleen. Figure 5D is a set of representative images showing one of these channels. Note that, although there are several adherent cells (green) in the image, only the moving cell is yellow. This is a result of the colocalization of the red transfluospheres with the green-labeled T cell.

The diameter of vessels in which T lymphocyte rolling occurred was compared between the MLN HEV and spleen. The mean ± SE diameter of the HEV in the MLN was 28.0 ± 1.0 µm; although similar in diameter to the PLN and Peyer's patch HEV, it was greater than twice the diameter of the channels in the spleen in which lymphocytes tethered, rolled, and adhered (11.6 ± 0.8 µm, P < 0.005) (49). In the spleen, it appeared that adhesion of T cells occurred within channels in the spleen parenchyma immediately distal to endothelium-lined terminal vessels. Interestingly, as shown in Table 1, these channels had diameters that were approximately twice that of the terminal vessels that fed them.

Comparison of lymphocyte trafficking behavior in the MLN and spleen. To determine whether T cell accumulation in the spleen occurred by a specific mechanism or was simply due to mechanical issues, such as filtering, we compared the rolling velocities of T cells and 3-µm-diameter beads. Because not all cells or beads that were observed appeared to interact with the vessel wall, we classified cells and beads on whether they contacted the vessel wall or not. Interacting cells or beads were those that did make contact, tethered, or adhered during passage through the visualized field. For adherent cells, the velocity reported is that observed just before adhesion.

As seen in Fig. 6, both beads and T cells traveled at a slower velocity before adhesion; however, T cells were traveling at about one-half the velocity of the beads [means ± SE: 154 ± 24 vs. 267 ± 18 µm/s, P = 0.01; median (interquartile range): 124 (83-227) vs. 223 (124-323) µm/s].


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  		Fig. 6.   Comparison of T lymphocyte and polymer velocities in the spleen and MLN. The mean (±SE) velocities (in µm/s) for interacting and noninteracting T cells or beads are plotted for the spleen and MLN. Note that no treatment had any effect on T lymphocyte rolling velocities in the spleen; however, T cells were noted to roll with a significantly lower velocity than the 3-µm-diameter inert polymer beads. No differences in the velocities were noted for noninteracting cells or beads in the spleen. In the MLN, the rolling velocity was significantly increased with either the loss of L-selectin or blockade of CD49d. This effect appeared to be additive, as the combination led to a significant increase in velocity over either intervention alone. No beads were noted to interact in the MLN HEV, and so no interacting velocity is given. Note that the noninteracting velocities were also increased with each of the treatments; however, none of the differences were statistically significant. Data from 3-6 mice were examined per condition.

A similar comparison of bead and T cell velocities was performed in the MLN HEV. As expected, given the known adhesive paradigm for the MLN HEV, no beads were noted to roll or interact with the endothelium. Velocities of noninteracting T cells and beads were similar, as shown in Fig. 6.

With the use of T lymphocytes from a wild-type host or an L-selectin-deficient host treated with either a nonspecific IgG or an anti-CD49d monoclonal antibody (anti-alpha 4 integrin), we calculated the effects that these various molecules had on the velocity at which T lymphocytes moved through the MLN or spleen. Figure 6 compares the resulting velocities of T lymphocytes in the MLN and spleen. Although the data presented are for mean velocities (±SE), similar results were obtained by comparing median values. The velocity of interacting T cells was significantly slower in the MLN [mean ± SE: 60 ± 8 µm/s; median (interquartile range): 38 (23-78) µm/s] compared with the spleen [mean ± SE: 154 ± 24 µm/s; median (interquartile range):124 (83-227) µm/s].

As shown in Fig. 6, blocking CD49d or a lack of L-selectin on the lymphocyte leads to a significant increase in interacting velocities [means ± SE: 514 ± 54 and 476 ± 19 µm/s; medians (interquartile range): 540 (274-712) and 421 (325-619) µm/s, respectively]; however, the loss of function of both molecules led to the greatest velocity increase [mean ± SE: 1,180 ± 70 µm/s; median (interquartile range): 1,176 (773-1,583) µm/s]. This is similar to the velocity of cells in the blood and likely is too rapid to allow for any interaction within the MLN.

When we examined the role of L-selectin and CD49d in the spleen, as shown in Fig. 6, there was no effect on the velocity of interacting cells with blockade of CD49d on L-selectin-deficient T cells. Therefore, we found no evidence that these molecules supported preadherent or adherent interactions in T cell trafficking within the white pulp of the spleen.

Velocity frequencies. Perhaps a more useful way to examine the effect of L-selectin and CD49d on the velocities of T lymphocytes in the MLN and spleen is to study the frequency of their respective velocities. This was done by dividing the velocities into classes, starting with 0-99 µm/s and increasing at 100 µm/s intervals. As can be seen from Fig. 7, wild-type T lymphocytes tend to travel in the MLN at velocities under 100 µm/s, as has been shown in the PLN and Peyer's patches. However, with the blockade of CD49d, velocities increase but remain under 1,000 µm/s. Loss of L-selectin led to a greater increase in velocity. Finally, loss of both CD49d and L-selectin lead to an increase of velocity to >1,200 µm/s. This is a speed clearly at which little interaction is able to take place between the cells and HEV.


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  		Fig. 7.   Comparison of T cell and polymer bead velocity classes in the MLN. Velocities were divided into 100 µm/s classes, and the percentage of cells or beads with a given velocity in the first 5 min after intravenous transfer (see METHODS) was plotted. A: velocity classes of wild-type cells. As expected, most cells traveled at speeds under 100 µm/s. B: effect of L-selectin-deficient T lymphocytes on the velocity classes. The velocities are increased, and no cells are capable of rolling under 200 µm/s. C: effect of an anti-CD49d blocking monoclonal antibody. Unlike in B, some cells (~8%) are still capable of rolling at speeds under 200 µm/s, presumably on L-selectin. D: effect of blocking CD49d in L-selectin-deficient T lymphocytes. There is a large shift in velocities, with most cells traveling at speeds >1,200 µm/s. E: 3-µm-diameter polymer beads do not roll or interact within the MLN HEV. Note, however, that the beads traveled at velocities somewhat lower than the anti-CD49d monoclonal antibody-treated L-selectin-deficient T lymphocytes. n >=  3 for all conditions.

Figure 8 shows the result of a similar evaluation of velocities in the spleen. Compared with the MLN, there is a larger spread of velocity classes using wild-type T lymphocytes, although there is still a significant fraction of cells moving at speeds under 100 µm/s. However, unlike the MLN, in the spleen nearly one-half of the cells were found to have a velocity of 300-700 µm/s. In the MLN, similar velocities could be attained only by interfering with either L-selectin or CD49d. However, as seen in Fig. 8, B and C, little effect was found using L-selectin-deficient T cells alone or with blockade of CD49d in the spleen. Indeed, if anything, there may have been a slight (not statistically significant) slowing of velocities in these two interventions.


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  		Fig. 8.   Comparison of T cell and polymer bead velocity classes in the spleen. Velocities were divided into 100 µm/s classes, and the percentage of cells or beads with a given velocity in the first 5 min after intravenous transfer (see METHODS) was plotted. A: velocity classes of wild-type cells. Most cells traveled at speeds under 700 µm/s, and a significant portion moved at <100 µm/s. B: effect of using L-selectin-deficient T lymphocytes. There was little, if any, change in the velocities of the cells, although fewer cells appeared to be traveling at speeds >500 µm/s. Similarly, little effect was seen on the velocity with blocking CD49d in L-selectin-deficient T lymphocytes, as shown in C. Similarly, the vast majority of 3-µm-diameter polymer beads (D) were noted to move through the spleen at velocities under 500 µm/s. n >=  3 for all conditions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The spleen is the largest of the secondary lymphoid organs and is the major site of immune responses against blood-borne antigens. Whereas data exist on the chemokines involved in separating T cells from B cells in the spleen, there is scant information on the mechanism by which lymphocytes exit the blood and enter the lymphoid compartment of the spleen (2, 14, 15). In an attempt at clarifying these events, we compared the movement of T lymphocytes in the spleen with the MLN, which expresses similar adhesion molecules as those found in the spleen. While the MLN HEV was easy to identify because of its size and the activities of lymphocytes within its lumen, the spleen did not have similar signature locations at which lymphocytes demonstrated behavior consistent with interaction between the lymphocyte and microvascular endothelial cells. It has been assumed that the MAdCAM-1-expressing marginal sinus was the site at which these interactions took place (20). However, we saw no transferred cells appearing or rolling at the periphery of the white pulp, which is contrary to what would be expected if the marginal sinus were the initial site of entry into the white pulp. Indeed, in most cases, the cells appeared at the center of the white pulp and moved toward the periphery, suggesting that entry into the white pulp occurs within channels located well within the lymphoid compartment of the spleen. Indeed, in many cases, numerous cells would appear and move through a single channel within a brief (30 s) time point. Then, for several minutes, no additional cells would appear to use this initial vascular channel, and, instead, another "hotspot" would appear, where cells entered into the lymphoid compartment of the spleen. The pulsating vessels, which may be the arteriolar capillaries that were reported in human spleen, control the flow of blood into the white pulp nodules and may be responsible for these hotspots (10). In fact, a recent report showed that blood flow to the rat spleen could be greatly increased by denervation of the organ, suggesting the existence of a mechanism for control of blood flow in the spleen (35). It is quite feasible that the autonomic nervous system controls these pulsating vessels allowing for rapid changes in blood flow through the spleen. Therefore, these pulsating vessels may be important in controlling the quantity of blood-borne antigens circulating through individual white pulp nodules and thus are important in the filtering capacity of the spleen.

Immediately distal to the pulsating vessels are the terminal vessels, which we suspect are arteriolar capillaries that end upon the honeycomb channels, which are composed of extracellular matrix proteins. In our estimation, these channels are within the white pulp and not at the marginal sinus, as shown by localization of transfluospheres compared with TEK expression and marginal sinus markers. Traditionally, the marginal sinus is thought to be lined by endothelial cells (36, 37). However, these honeycomb/bud channels show no expression of TEK, and we interpret this to indicate that they lack an endothelial lining. We acknowledge, however, that there are rare subpopulations of endothelial cells that are TEK- and that our current experiments cannot exclude the presence of such TEK- endothelial cells in this interesting compartment. Our interpretation that there are no endothelial cells in this compartment is based on the morphological picture of blood oozing into the buds at this tissue location, the lack of TEK expression, and the change in diameter of the channels in the honeycomb region, all of which suggest that this compartment is defined simply by channels consisting of extracellular matrix proteins. It is unclear whether these honeycomb regions are part of the marginal sinus, although hemodynamic data (Table 1) would suggest that they are sinus like. However, in casts of the murine spleen, the marginal sinus structures were 5- to 10-µm-diameter well-delineated structures (38). The honeycomb regions we see appear to be larger than this and do not have a well-defined TEK+ endothelial border. Indeed, these structures seem more akin to the perifollicular zone recently reported in the human spleen (42). "Terminal vessels" were found to end on fibroblast-lined channels that lacked an endothelial layer at the juncture of the white and red pulp. These structures were not seen in corrosion casts of the spleen, and it was assumed that this was due to the digestion process during the casting procedure. If this is the case, it is possible that a similar perifollicular structure may occur in the mouse and may be the honeycomb region in which we see cells firmly adhering. Clearly this structure is within the white pulp, as cells begin rolling from within the transfluosphere region and firmly adhere near its terminus. Figure 9 is a schematic of our current understanding of the vascular entry into the white pulp. Further characterization of this novel tissue compartment will provide important insights into the factors governing leukocyte behavior in cells newly entering the splenic white pulp both under homeostatic conditions and in the setting of microbial infection or systemic inflammatory diseases.


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  		Fig. 9.   Schematic diagram of T lymphocyte entry into the white pulp. Blood enters the central arteriole from large arterial vessels. On the basis of our data, the blood then enters pulsating vessels (indicated by the dark gray ovals suggesting a muscular sheath). These vessels then divide into smaller terminal vessels, which give rise to the honeycomb-like regions of the white pulp, which lack a TEK-expressing endothelial layer. It is unclear from our data whether these regions are part of the marginal sinus, as in A, or are closer to the center of the lymphoid nodule, as shown in B. The gray arrowheads indicate the path of T cells. The draining of blood from these honeycomb areas is unclear and is indicated by the dashed lines and question marks.

Our hemodynamic data suggest that specific carbohydrate (selectin)-based interactions could be possible between migrating lymphocytes and the spleen microvasculature. WSRs in the range of 220-330 s-1 are reported for PLN HEV and rabbit mesentery venules, with slightly higher values reported in the rat mesentery and Peyer's patches (491 ± 7 and 416 ± 52 s-1, respectively) (30, 31, 45, 49). Similar WSRs were obtained in the terminal vessels, suggesting that selectin (or alpha 4-integrin)-mediated rolling interactions could occur in these vessels. In the honeycomb/bud region, however, most cell movement was due to a significant mechanical interaction, as Vblood in these "vessels" was similar to that at which the lymphocytes were moving. In addition, in this region, the WSR/WSS values are quite low but still capable of supporting adhesive events, because they are similar to the values reported for sinusoids and intermediate venules of the bone marrow (28-30). Indeed, the hemodynamic data are consistent with the idea that the honeycomb/bud region is indeed a sinus-like structure, although, as mentioned above, it is unclear whether this region is part of the marginal sinus.

Cellular rolling velocities were calculated before and after adhesion molecule blockade to determine whether CD49d or L-selectin plays any role in migration to the spleen. Both of these molecules have been reported to be important in the MLN; however, neither has ever been shown to be important in the spleen (although, as mentioned previously, no adhesion molecules have ever been associated with a loss of splenic cellularity) (3, 5, 9, 20-23, 27, 39, 43, 47). The unimpeded T cell velocities reported in the MLN are similar to those reported for PLN and Peyer's patch HEV; however, values obtained in the spleen were much higher, but were still within reported values for speeds achieved on L-selectin-expressing transfectants in vitro (~181 ± 6.9 µm/s), but higher than that reported in vivo (25-60 µm/s) (16, 41, 49).

With the use of L-selectin-deficient T lymphocytes with or without CD49d blockade, we were unable to alter the rolling velocities or adhesion of cells in the spleen. Therefore, we conclude that these adhesion molecules play little, if any, role in homing to the spleen. Indeed, we speculate that the majority of the adhesive events in the spleen are due to mechanical interactions, possibly controlled by the autonomic nervous system (via the pulsating vessels); however, further investigations will be needed to verify this assertion. Although our data were not revealing for the spleen, they do give some insight into the relative importance of these molecules in the MLN. If we presume that without CD49d all interactions between the HEV and T cells occur via L-selectin, then our data suggest that rolling on L-selectin occurs at velocities in the range of 100-800 µm/s, which is supported by the reported in vitro data (13). Similarly, if we assume that without L-selectin all rolling in the MLN occurs via CD49d, then this interaction produces velocities in the range of 300-1,500 µm/s. Furthermore, the lack of any cells rolling with a velocity under 200 µm/s in the L-selectin-deficient group indicates that CD49d-mediated rolling of T lymphocytes in the MLN does not occur at these low velocities.

In conclusion, these data show that, although both organs contain MAdCAM-1-expressing cells, T lymphocyte homing to the spleen and MLN occurs via different mechanisms. The differences and similarities between secondary lymphoid organs are shown in Table 2. Rolling in the MLN HEV is dependent on both CD49d and L-selectin, possibly with CD49d mediating more rapid rolling and L-selectin being responsible for a slower velocity of interaction. Abrogation of both adhesion molecules led to a significant increase in lymphocyte velocity in the MLN. This is similar to what has been reported in Peyer's patches, the organ with which the MLN has the most in common. Movement of lymphocytes into the spleen, however, was unaffected by blocking L-selectin or CD49d, even though the terminal vessels had hemodynamic properties similar to the MLN HEV. Therefore, our data show that neither of these molecules plays a dominant role in T cell rolling in the spleen. Furthermore, our data indicate that a significant component of T cell adhesion in the spleen is likely due to mechanical effects.

                              
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  		Table 2.   Comparison of T lymphocyte migration characteristics in secondary lymphoid organs

We have further defined the vasculature of the spleen, showing that blood flow initially goes from pulsating vessels into terminal vessels and finally into terminal buds or honeycombs. In the terminal bud region, channels are found that lack a TEK-expressing endothelial cell lining, and this is where lymphocytes firmly adhered. On the basis of hemodynamic values, however, we postulate that rolling interactions of lymphocytes take place in the terminal vessels, immediately proximal to the buds. Although L-selectin and CD49d are important in the migration of T lymphocytes into the MLN HEV, these adhesion molecules are not involved in rolling and adherence of T lymphocytes in the vascular channels of the white pulp of the spleen. Our data indicate that T cells firmly adhere in the honeycomb/bud region of the splenic vasculature. While this region has a WSR similar to bone marrow sinusoids, it is unclear whether it does represent the marginal sinus. Specifically, the lack of TEK-expressing endothelium and the larger vessel size suggest that this discrete structure is not the marginal sinus, which separates the white pulp from the red pulp. Further investigations are being directed at better understanding this unique compartment, which is not found in other secondary lymphoid organs.


    ACKNOWLEDGEMENTS

We thank Dale F. Osborne and Michelle Rohlfing for excellent technical assistance, Carlene Zindl for helpful discussions, and Dr. Charles Parker for the critical review of the manuscript.


    FOOTNOTES

This work was supported by a Barnes-Jewish Foundation grant (to M. H. Grayson), National Institutes of Health Grants AI-01800 (to M. H. Grayson), AI-34580 (to D. D. Chaplin), and GM-44118 (to R. S. Hotchkiss), and the Alan A. and Edith L. Wolff Foundation.

Address for reprint requests and other correspondence: M. H. Grayson, Washington Univ. School of Medicine, Campus Box 8122, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: wheeze{at}allergist.com).

1 The on-line version of this article contains supplemental material (see http://ajpheart.physiology.org/cgi/content/full/284/6/H2213/DC1).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 13, 2003;10.1152/ajpheart.00999.2002

Received 18 November 2002; accepted in final form 4 February 2003.


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