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International Immunology Advance Access originally published online on January 30, 2007
International Immunology 2007 19(3):321-329; doi:10.1093/intimm/dxl149
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

P-selectin glycoprotein ligand-1 mediates L-selectin-independent leukocyte rolling in high endothelial venules of peripheral lymph nodes

Nari Harakawa1,2, Akiko Shigeta3, Masahiro Wato4, Glenn Merrill-Skoloff5, Barbara C. Furie5, Bruce Furie5, Toshiro Okazaki2, Naochika Domae1, Masayuki Miyasaka6 and Takako Hirata3

1 Department of Internal Medicine, Osaka Dental University, Hirakata, Osaka, Japan
2 Division of Clinical Laboratory Medicine/Hematology, Faculty of Medicine, Tottori University, Yonago, Tottori, Japan
3 The 21st Century Center of Excellence Program, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
4 Department of Oral Pathology, Osaka Dental University, Hirakata, Osaka, Japan
5 Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA
6 Laboratory of Immunodynamics, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan

Correspondence to: T. Hirata; E-mail: thirata{at}biken.osaka-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Lymphocyte homing to peripheral lymph nodes (LNs) requires L-selectin. Previous studies, however, suggest that there are L-selectin-independent mechanisms of lymphocyte homing. P-selectin glycoprotein ligand-1 (PSGL-1) is a major ligand for P-selectin expressed in a selectin-binding form on myeloid cells and subsets of lymphoid cells. To discover whether PSGL-1 plays a role in lymphocyte homing, we examined leukocyte rolling and adhesion in the high endothelial venules (HEVs) of the subiliac LNs of wild-type and PSGL-1-deficient mice by intravital microscopy. There were no significant differences in blood velocity or wall shear stress between wild-type and PSGL-1-deficient mice. Although the leukocyte rolling fraction was not altered in PSGL-1-deficient mice, infusion of an anti-L-selectin mAb into these mice completely abolished leukocyte rolling, while the same treatment in wild-type mice inhibited 90% of the leukocyte rolling. This residual rolling in wild-type mice appears to depend on the PSGL-1–P-selectin interaction, since infusion of an anti-L-selectin mAb together with an anti-PSGL-1 mAb or anti-P-selectin mAb almost completely abolished the rolling. PSGL-1 deficiency also led to a higher rolling velocity, suggesting that PSGL-1 mediates leukocyte rolling at low velocities. P-selectin was found to be expressed on the HEVs of subiliac LNs under the conditions of intravital microscopy. Taken together, these results indicate that the interaction of PSGL-1 with P-selectin constitutes a second mechanism of leukocyte rolling in the HEVs of peripheral LNs.

Keywords: adhesion molecules, cell trafficking, inflammation, intravital microscopy, lymphocytes


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The recirculation of lymphocytes is crucial for the development of the immune response to foreign antigens. Lymphocytes migrate from the blood into peripheral lymph nodes (peripheral LNs; PLNs) through specialized post-capillary venules called high endothelial venules (HEVs), where lymphocytes tether and roll via L-selectin (CD62L), a member of the selectin family of adhesion receptors expressed on most leukocytes (1–3). The ligands for L-selectin presented on HEVs consist of a group of sulfated sialoglycoproteins, collectively termed the peripheral node addressin (PNAd) (4, 5). Some rolling lymphocytes firmly attach to the endothelium through the activation-induced engagement of the ß2 integrin leukocyte function-associated antigen (LFA)-1, and subsequently emigrate from the venule into the underlying parenchyma. Studies using blocking antibodies or gene-targeted mice have demonstrated the absolute requirement for L-selectin in lymphocyte recruitment to PLNs (6–10). However, intravital microscopy of the HEVs in PLNs showed that even in L-selectin-deficient mice or after blockade with the anti-L-selectin mAb MEL-14, some circulating leukocytes could still roll, suggesting the existence of additional cell adhesion interactions besides those mediated by L-selectin (11–13).

Naive lymphocytes, which express L-selectin at high levels, are the major population to enter the PLNs via the HEVs (14, 15). In contrast, memory lymphocytes typically enter the PLNs via afferent lymphatics, although some do appear to enter the PLNs via the HEVs (16, 17). The recruitment of memory lymphocytes to the PLNs is also partly inhibited by the MEL-14 mAb, suggesting that these lymphocytes use L-selectin-dependent mechanisms (17). Most memory lymphocytes, however, are L-selectinlow and show high expression of several adhesion molecules, such as CD44 and LFA-1 (14, 18, 19). Whether memory lymphocytes use adhesion molecules other than L-selectin to tether and roll in HEVs remains unknown.

P-selectin glycoprotein ligand-1 (PSGL-1; CD162) is a sialomucin that was identified as the major ligand for P-selectin (CD62P) on myeloid cells and subsets of lymphoid cells (20). PSGL-1 also functions as an E-selectin (CD62E) ligand (21, 22). Both P- and E-selectin are expressed on the endothelial cells of post-capillary venules during inflammation (23), and PSGL-1 thus mediates leukocyte rolling in inflamed post-capillary venules via interactions with endothelial P- and E-selectin. In addition, PSGL-1 functions as an L-selectin ligand to mediate interactions between leukocytes (24–27). PSGL-1 on rolling or adherent leukocytes or on leukocyte fragments has been shown to mediate the L-selectin-dependent leukocyte rolling observed in inflamed post-capillary venules (28). To bind selectins, PSGL-1 requires specific core 2-type O-glycans containing the sialyl LewisX moiety. The expression of some glycosyltransferases involved in the synthesis of selectin-binding glycans, including core 2 ß-1,6-N-acetylglucosaminyltransferase and {alpha}-1,3-fucosyltransferase, is dynamically regulated in lymphocytes during activation and differentiation (29–31). Thus, although most lymphocytes express PSGL-1, only particular subsets of lymphocytes, notably subsets of memory and effector T cells, express a form of PSGL-1 that can bind selectins. The role of PSGL-1 in the recruitment of a subset of memory/effector T cells to sites of inflammation has been demonstrated (21, 32). Whether PSGL-1 plays any role in lymphocyte recruitment to the PLNs, either under steady state or inflammatory conditions, has not been studied.

To evaluate the role of PSGL-1 in leukocyte rolling in the PLNs, we studied leukocyte rolling in the subiliac LNs of mice lacking PSGL-1. Here, we demonstrate that PSGL-1 is not required for leukocyte rolling in the HEVs of the PLNs. However, a PSGL-1 interaction with P-selectin mediates the L-selectin-independent rolling of leukocytes in the HEVs. These results may suggest a novel function for PSGL-1 in lymphocyte trafficking to the PLNs under certain inflammatory conditions in which P-selectin is expressed.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
PSGL-1-deficient (PSGL-1–/–) mice, prepared by gene targeting, have been previously described (21, 33). Twelve- to 16-week-old PSGL-1–/– mice (20–30 g body weight) from a fifth generation back-cross into a C57BL/6 background were used. Control C57BL/6 mice (20–30 g body weight) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were housed in the animal facilities of Beth Israel Deaconess Medical Center and in the Institute of Experimental Animal Sciences at Osaka University Medical School. All studies and procedures were approved by the Animal Care and Use Committee of Beth Israel Deaconess Medical Center and by the Ethics Review Committee for Animal Experimentation of the Osaka University Graduate School of Medicine.

Reagents
Rhodamine 6G was purchased from Molecular Probes (Eugene, OR, USA). GM 6001 was purchased from Calbiochem (La Jolla, CA, USA). Neutralizing mAbs MEL-14 (rat IgG2a, anti-mouse L-selectin), 2PH1 (rat IgG1, anti-mouse PSGL-1), RB40.34 (rat IgG1, anti-mouse P-selectin) and MECA-79 (rat IgM, anti-PNAd) were purchased from BD Biosciences (San Jose, CA, USA).

Flow cytometry
All mAbs used for flow cytometric analyses were purchased from BD Biosciences. They included FITC–anti-CD4 (RM4-5), FITC–anti-CD8 (53-6.7), FITC–anti-CD44 (IM7), PE–anti-L-selectin (MEL-14), PE–anti-CD45RB (16A), allophycocyanin (APC)–anti-CD4 (RM4-5) and APC–anti-CD8 (53-6.7). P-selectin-binding activity was examined by flow cytometry as described previously (34) except that P-selectin-IgG (BD Biosciences) was used instead of P-selectin-IgM. Leukocytes from the peripheral blood, PLNs and spleen of wild-type and PSGL-1–/– mice were incubated with 10 µg ml–1 P-selectin-IgG for 30 min on ice, washed, and then incubated with biotinylated anti-human IgG (American Qualex, San Clemente, CA, USA). The cells were then washed and incubated with APC–streptavidin or PE–Cy5–streptavidin (both from BD Biosciences) with or without the mAbs indicated above. Non-specific staining was determined by the addition of 10 mM EDTA. To prevent L-selectin shedding, 100 µM GM 6001 was added to the incubation buffer throughout the experiment. The cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). For sorting of naive and memory CD4+ T cells, cells were stained with FITC–anti-CD44, PE–anti-CD45RB and APC–anti-CD4. Naive (CD44lowCD45RBhigh) and memory (CD44highCD45RBlow) CD4+ T cells were sorted on a FACSVantage SE (BD Biosciences).

Determination of peripheral blood leukocyte number and differential counts
Whole blood was obtained from the tail vein before and after injection of 50 µg of an anti-L-selectin, anti-PSGL-1, anti-P-selectin or anti-PNAd mAb. The blood smear was stained with Wright–Giemsa stain. The systemic leukocyte counts were determined using a hemocytometer.

Subiliac LN preparation and visualization by intravital microscopy
Mice were pre-anesthetized with an intra-peritoneal injection of 125 mg kg–1 of ketamine HCL (Parke Davis, Detroit, MI, USA), 12.5 mg kg–1 xylazine (Phoenix Pharmaceuticals, St Joseph, MO, USA) and 0.25 mg kg–1 atropine sulfate (American Reagent Laboratories, Shirley, NY, USA). The trachea was intubated to facilitate spontaneous respiration. To maintain anesthesia and neutral fluid balance, sodium pentobarbital (Abbott Laboratories, North Chicago, IL, USA; 5 mg ml–1 in saline) was administered in volumes of 20–40 µl through the cannulated jugular vein. In experiments using neutralizing mAbs, mice received 50 µg of mAb injected through the jugular cannula. The left subiliac LNs were dissected as described by von Andrian (35). The node was exteriorized onto an intravital microscopy tray and superfused with thermocontrolled (37°C) and aerated (5% CO2, 95% N2) bicarbonate-buffered saline throughout the experiment. The HEVs were observed by intravital microscopy using an Axioskop (Carl Zeiss, Thornwood, NY, USA) fitted with an Achroplan (40x, 0.80 numerical aperture) water-immersion objective, long-distance condenser and a stabilized DC power supply. Leukocyte rolling in the venules was recorded for 70–120 s using a CCD camera (SSC-S20; Sony, Tokyo, Japan) connected through an IBM-compatible computer to a Sony SVHS video recorder (SVA-9500MD).

Analysis of leukocyte rolling in HEVs of subiliac LNs
Circulating leukocytes were labeled by intravenous injection of rhodamine 6G (1 mg ml–1 in saline, 10 ml kg–1). Myeloid and lymphoid cells and platelets were visualized. Video recordings from the intravital microscopy experiments were analyzed on a computer-based image acquisition system and vessel diameter was determined using Scion Image. The mean blood flow velocity (Vblood) was calculated from the velocity measurements of polystyrene microspheres in each venule according to the relationship: Vblood = Vmax/[2- (DM/DV)2], where DM is the microsphere diameter and DV the vessel diameter. The volumetric blood flow, wall shear rate, critical velocity and rolling velocity (Vroll) were calculated as described by von Andrian et al. (36) and Yang et al. (33). To correct for the influence of the varying Vblood upon rolling, the relative rolling velocity (Vrel) was calculated according to the following formula: Vrel = Vroll/Vblood x 100%. Pooled Vroll data are a sensitive indicator of the strength of the rolling interaction (35). The total leukocyte fraction was determined as the product of the measured systemic leukocyte concentration and blood flow in the venule. To compensate for differences in the systemic leukocyte counts, the rolling behavior of leukocytes in the LN vessels is presented as the rolling leukocyte fraction; the number of rolling leukocytes in the vessel as a percentage of the total leukocyte fraction. The sticking fraction shows the percentage of interacting cells that became immobile for 30 s.

Immunohistochemistry
Subiliac LNs were removed after exteriorization, fixed in formalin and embedded in paraffin. The paraffin blocks were cut into 4-µm sections. The sections were deparaffinized by ethanol and d-limonene and then immersed in 3% H2O2 to block endogenous peroxidase. The sections were incubated with goat anti-P-selectin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 30 min, then with peroxidase-conjugated anti-goat IgG (Histofine simple stain MAX-PO(G), Nichirei Biosciences, Tokyo, Japan) at room temperature for 30 min, and visualized with 3,3'-diaminobenzidine (DakoCytomation, Carpinteria, CA, USA) at room temperature for 5 min. The sections were counterstained with hematoxylin and observed by light microscopy.

In vivo labeling of P-selectin
The fluorescent-carboxylated YG microspheres (1-µm diameter) coated with protein G were purchased from Polysciences (Warrington, PA, USA). Thirty minutes before the observation of subiliac LNs, 1 x 108 per 100 µl of protein G-coupled beads were mixed with 50 µg of either an irrelevanat rat IgG1 (BD Biosciences) or the anti-P-selectin mAb RB40.34. The mixture was allowed to incubate for 30 min before the beads were re-suspended in isotonic saline with 1% FCS and vortexed to break any aggregates. The mice were injected with three sequential 50 µl aliquots of rat IgG1-coupled beads >5 min followed by a 10-min resting period to allow for clearance of circulating beads. Subsequently, the RB40.34-coupled beads were injected in the same manner, and bound beads were observed by intravital fluorescent microscopy.

Statistical Analysis
Data are presented as the mean ± SD. A two-tailed Student's t-test was used for the statistical comparison of two samples, when applicable. Multiple comparisons were performed by the Kruskal–Wallis test and Bonferroni correction of the P-value. Histograms of rolling velocity (% of Vblood) were compared using the Mann–Whitney U-test. Differences were considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
PSGL-1 deficiency does not affect the leukocyte rolling fraction in subiliac LN HEVs
Although previous work has clearly demonstrated the specialized role of L-selectin in leukocyte rolling in the HEVs of PLNs, the existence of additional cell adhesion interactions besides those mediated by L-selectin has also been suggested (11–13). To evaluate the potential role of PSGL-1 in leukocyte rolling in the HEVs of PLNs, we compared leukocyte rolling in a subiliac LN of wild-type and PSGL-1–/– mice. The observations were made by intravital microscopy of the dissected subiliac LN of an anesthetized mouse. As reported previously (35), five branching orders (I through V) can be identified in a typical venular tree in the subiliac LN. Lower-order venules are formed by the confluence of higher-order venules. Venules of orders III through V have the properties of HEVs. As shown in Table 1, we did not observe significant differences in mean blood flow velocity (Vblood), wall shear rate, or blood flow between wild-type and PSGL-1–/– mice in venules belonging to any order. There were also no significant differences in vessel diameter, except in order V venules, which were smaller in PSGL-1–/– mice than in wild-type mice. Overall, there were no hemodynamic differences between wild-type and PSGL-1–/– mice.


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  		Table 1. Properties of the post-capillary venules in subiliac LNs

 
To quantify the leukocyte–vessel wall interactions in subiliac LNs, rhodamine 6G-stained endogenous leukocytes were observed by intravital microscopy. In both wild-type and PSGL-1–/– mice, the rolling fractions of leukocytes in order I and II venules were significantly lower than in order III through V HEVs (Fig. 1A). There were no significant differences in the rolling leukocyte fractions in the different venule orders between PSGL-1–/– and wild-type mice.


Figure 1
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  		Fig. 1. Leukocyte rolling and sticking fractions in venules of orders I through V in the subiliac LNs of wild-type and PSGL-1–/– mice. Leukocyte rolling fractions (A) and sticking fractions (B) were measured as described in Methods. Data shown are the mean ± SD of results from seven animals (7–11 venules each). Differences in rolling and sticking fractions between venules of orders I–II and orders III–V were statistically significant (*P < 0.05).

 
In both mouse genotypes, a sub-population of rolling leukocytes adhered firmly to the vessel wall. Like the rolling fractions, the sticking fractions were also significantly higher in order III–V HEVs than in order I and II venules in both wild-type and PSGL-1–/– mice (Fig. 1B). There were no significant differences in the sticking leukocyte fractions in each venule order between wild-type and PSGL-1–/– mice. These data suggest that PSGL-1 may not play a significant role in leukocyte rolling and subsequent sticking in the HEVs of subiliac LNs or that its role may be masked by the dominant function of L-selectin, which plays a critical role in leukocyte rolling in the HEVs of PLNs.

L-selectin-independent leukocyte–HEV interaction is mediated by PSGL-1
Since L-selectin is the major adhesion molecule mediating leukocyte–HEV interactions in PLNs, we next compared leukocyte–HEV interactions in PSGL-1–/– and wild-type mice in the presence and absence of a blocking anti-L-selectin mAb MEL-14. Leukocyte rolling was measured in order III HEVs before and after infusion of MEL-14. Infusion of MEL-14 did not significantly change the number of circulating leukocytes (data not shown). Leukocyte rolling was inhibited to 10% of the original value by the infusion of MEL-14 into wild-type mice (Fig. 2A, left panel). In contrast, infusion of MEL-14 into PSGL-1–/– mice caused almost complete inhibition of leukocyte rolling (Fig. 2A, right panel). The combined infusion of MEL-14 and an anti-PSGL-1 mAb 2PH1 almost completely inhibited leukocyte rolling (Fig. 2A, left panel). Leukocyte sticking was also only 41% of the control, following infusion of MEL-14, and it was further inhibited in wild-type mice by the infusion of MEL-14 and 2PH1 combined (Fig. 2B, left panel). In contrast, the infusion of MEL-14 alone into PSGL-1–/– mice inhibited leukocyte sticking to the level obtained in wild-type mice with the combined MEL-14 and 2PH1 blockade (Fig. 2B, right panel). These results suggest that PSGL-1 is a component of a second mechanism for leukocyte rolling and sticking in HEVs in subiliac LNs.


Figure 2
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  		Fig. 2. L-selectin-independent leukocyte rolling and sticking in order III venules of the subiliac LNs is mediated by PSGL-1. Leukocyte rolling fractions (A) and sticking fractions (B) were analyzed before and after the treatment of wild-type and PSGL-1–/– mice with the blocking anti-L-selectin mAb, MEL-14. Wild-type mice were also treated with a combination of MEL-14 and the anti-PSGL-1 mAb 2PH1 or the anti-P-selectin mAb RB40.34. Data shown are the mean ± SD of results from seven and eight experiments for wild-type and PSGL-1–/– mice, respectively. *P < 0.05 compared with control (before treatment with the indicated mAbs).

 
Since PSGL-1 is the predominant ligand for P-selectin, we next used an anti-P-selectin mAb to assess the contribution of P-selectin to leukocyte rolling in the subiliac LN of wild-type mice. The combined infusion of an anti-P-selectin mAb, RB40.34, and the anti-L-selectin MEL-14 completely inhibited leukocyte rolling (Fig. 2A, left panel) and nearly completely inhibited leukocyte sticking (Fig. 2B, left panel). These results suggest that P-selectin is also a component of a second mechanism for leukocyte rolling. The similar effect of the anti-P-selectin mAb and the anti-PSGL-1 mAb suggests that the PSGL-1 interaction with P-selectin is likely to mediate the L-selectin-independent component of leukocyte rolling in HEVs.

PSGL-1 deficiency changes the rolling velocity of leukocytes in HEVs
The lack of adhesion molecules involved in leukocyte rolling may change not only the number of rolling leukocytes but also their rolling velocity (37, 38). P-selectin has been reported to mediate leukocyte rolling at lower velocities than L-selectin in the venules of the mouse cremaster muscle in vivo (39). We compared the velocities of rolling leukocytes in wild-type and PSGL-1–/– mice and found that they rolled at a higher velocity in the PSGL-1–/– mice, suggesting that PSGL-1 mediates leukocyte rolling at low velocities (Fig. 3A). The relative leukocyte velocity in the HEVs of wild-type mice also increased following the injection of the anti-P-selectin mAb RB40.34, parallel to the effect of PSGL-1 deficiency (Fig. 3B). These results suggest that, as in the venules of the mouse cremaster muscle (39), in the HEVs of the subiliac LN, the PSGL-1–P-selectin interaction mediates leukocyte rolling at lower velocities than does the L-selectin-mediated interaction.


Figure 3
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  		Fig. 3. Histograms of the relative rolling velocities of leukocytes in order III venules. (A) Histograms of relative rolling velocities in wild-type and PSGL-1–/– mice. A statistical comparison revealed that the rolling velocity of leukocytes in PSGL-1–/– mice was higher than in wild-type mice. (B) Histograms of the relative rolling velocities in wild-type mice before and after infusion of the anti-P-selectin mAb RB40.34. Data shown are the mean ± SD of results from seven experiments for each genotype. A statistical comparison revealed that the rolling velocity of leukocytes increased in wild-type mice after the infusion of RB40.34.

 
P-selectin is expressed in PLN HEVs
As the anti-P-selectin mAb inhibited L-selectin-independent leukocyte rolling in PLN HEVs, we examined whether P-selectin is expressed in HEVs. Immunohistochemistry of the subiliac LN failed to detect P-selectin expression in HEVs either before or after surgical stimulation, while an LPS-stimulated LN showed a strong staining for P-selectin in HEVs (data not shown). Thus we used a bead-based assay, which was shown to be a more sensitive assay to detect P-selectin expression (38). Fluorescent beads coated with the anti-P-selectin mAb RB40.34 were injected into the mouse in which the subiliac LN had been dissected for intravital microscopy. RB40.34-coated beads bound in much greater numbers to the order II and III venules compared with control beads coated with isotype control rat IgG (Fig. 4A). Binding was specific as injection of RB40.34 into the mice before injection of the beads reduced the binding of the RB40.34-coated beads to the background level (Fig. 4B). Similar binding of RB40.34-coated beads to the HEVs was observed in the PSGL-1–/– mouse (data not shown). These data suggest that P-selectin is indeed expressed on the surface of HEVs of subiliac LNs under surgical stimulation.


Figure 4
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  		Fig. 4. P-selectin expression in PLN HEVs. (A) Binding of anti-P-selectin-coated beads to the venules in subiliac LNs. One-micron fluorescent beads coated with control rat IgG or the anti-P-selectin mAb RB40.34 were injected and allowed to bind to the venules in subiliac LNs. Order II and III venules are shown. Bound beads are shown by arrowheads. (B) Inhibition of binding of anti-P-selectin-coated beads by pre-treatment of the mouse with the anti-P-selectin mAb. Pre-treatment of the mouse with RB40.34 prevented the binding of RB40.34-coated beads to the venules.

 
P-selectin-binding lymphocytes are enriched in the memory population
The results from the intravital microscopy study of the subiliac LNs suggested that L-selectin-independent leukocyte rolling in HEVs was mediated by a PSGL-1 interaction with P-selectin. Although PSGL-1 is expressed on most leukocytes, it requires appropriate post-translational modification to bind P-selectin. Therefore, we examined the P-selectin-binding activity of leukocytes from the peripheral blood and PLNs of wild-type and PSGL-1–/– mice by flow cytometry. As reported previously (20), all the granulocytes from wild-type mice bound P-selectin (data not shown), while only subsets of CD4+ and CD8+ T cells could bind it (Fig. 5A). Lymphocytes from PSGL-1–/– mice did not detectably bind P-selectin (Fig. 5A). The majority of P-selectin-binding CD4+ T cells had low L-selectin expression, although some expressed L-selectin at high levels (Fig. 5B). These P-selectin-binding cells were enriched in the CD44highCD45RBlow memory CD4+ T cell population, particularly in the population that expressed very high levels of CD44 (Fig. 5C). Similarly, CD8+ P-selectin-binding cells were enriched in the CD44high population (data not shown). These results suggest a possibility that PSGL-1 mediates the rolling of P-selectin-binding memory cells in the HEVs of PLNs.


Figure 5
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  		Fig. 5. P-selectin-binding activity on lymphocytes. (A) P-selectin-binding activity on lymphocytes from the peripheral blood and PLNs of wild-type and PSGL-1–/– mice, assayed by flow cytometry. Cell suspensions from the peripheral blood and PLNs of wild-type and PSGL-1–/– mice were incubated with P-selectin-IgG in the presence of calcium or EDTA. The cells were then incubated with biotinylated anti-human IgG followed by APC–streptavidin together with FITC–anti-CD4 or FITC–anti-CD8. (B) The expression of L-selectin and P-selectin-binding activity on CD4+ and CD8+ T cells from the peripheral blood of wild-type mice. Blood leukocytes were incubated with P-selectin-IgG. The cells were then incubated with biotinylated anti-human IgG followed by APC–streptavidin-, PE–anti-L-selectin and either FITC–anti-CD4 or FITC–anti-CD8. The expression of L-selectin and P-selectin-binding activity on gated CD4+ and CD8+ cells is shown. (C) P-selectin-binding activity on naive and memory CD4+ lymphocytes. Cell suspensions of PLNs from wild-type mice were stained with FITC–anti-CD44, PE–anti-CD45RB and APC–anti-CD4. Naive (CD44lowCD45RBhigh) and memory (CD44highCD45RBlow) CD4+ T cells were sorted on a FACSVantage SE. Sorted cells were stained with P-selectin-IgG followed by biotinylated anti-human IgG and PE–Cy5–streptavidin (open histograms). Non-specific binding was determined by the addition of 10 mM EDTA (filled histograms).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The specialized role of L-selectin in leukocyte–HEV interactions in PLNs is well established. L-selectin was originally identified as a homing receptor on lymphocytes, and its interaction with PNAd on HEVs has been extensively characterized. Although this L-selectin-dependent mechanism is required for lymphocyte homing and represents the dominant mechanism of leukocyte–endothelial interactions in HEVs, previous studies have reported ~10% residual leukocyte rolling in HEVs in the absence of L-selectin or upon inhibition of L-selectin function (11–13). We show that PSGL-1 interaction with P-selectin represents an alternate L-selectin-independent mechanism for rolling in the HEVs.

The PSGL-1 deficiency by itself had little effect on leukocyte rolling in the HEVs of PLNs. No differences in the numbers of rolling or sticking leukocytes were observed between wild-type and PSGL-1–/– mice. These data suggest that either PSGL-1 plays only a small role in leukocyte rolling and sticking in HEVs, or the lack of PSGL-1 can be compensated for by the expression of another molecule. In our experiments, the contribution of PSGL-1 to leukocyte rolling in HEVs was mostly unveiled only after the inhibition of L-selectin function by the anti-L-selectin mAb MEL-14. Our finding that the L-selectin-dependent mechanism played the dominant role in leukocyte rolling compared with the PSGL-1-dependent mechanism is supported by previous findings of structural abnormalities in the PLNs of the L-selectin-deficient mouse (7). The absence of L-selectin leads to significantly impaired leukocyte rolling and sticking in the HEVs and diminished lymphocyte influx into the PLNs, markedly reducing the size and cellularity of the PLNs. In contrast, we found the LN architecture and cellularity in the PSGL-1–/– mouse to be normal. This observation emphasizes that the PSGL-1-dependent pathway is not required for lymphocyte migration into the LN and cannot substitute for the L-selectin-dependent pathway.

Although it has been shown that the L-selectin-dependent leukocyte rolling observed in inflamed post-capillary venules is mediated by PSGL-1 expressed on already rolling or adherent leukocytes (28), our finding that the PSGL-1 deficiency had only a minor effect on leukocyte rolling in the HEVs of PLNs may suggest that PSGL-1 does not play a dominant role as an L-selectin ligand in HEVs. In accordance with previous reports (35), we observed considerable inhibition of leukocyte rolling after treating wild-type mice with the anti-PNAd mAb MECA-79 (data not shown), confirming that L-selectin-dependent rolling is mostly mediated via interactions with PNAd expressed on HEVs.

In this study, we did not distinguish between granulocytes and lymphocytes in our measurements of leukocyte rolling and sticking. Warnock et al. (13) previously demonstrated in a similar model that both lymphocytes and granulocytes roll in the HEVs, but only the lymphocytes stick to these venules. Therefore, our rolling experiments likely reflect the properties of both lymphocytes and granulocytes, while the sticking experiments primarily reflect the properties of lymphocytes. Since blocking mAbs inhibited both rolling and sticking, it is likely that inhibiting the rolling of lymphocytes and/or granulocytes leads to the subsequent inhibition of lymphocyte sticking. This result needs to be confirmed using purified populations of granulocytes and lymphocytes.

Among the leukocytes circulating in the blood, myeloid cells alone constitutively express functional PSGL-1, and only particular subsets of lymphoid cells express PSGL-1 in a selectin-binding form (40), particularly certain memory T cells. These cells may roll in HEVs under conditions in which P-selectin is expressed on the HEVs. P-selectin is not normally expressed on the surface of endothelial cells, but it is translocated to the cell surface in response to inflammatory stimuli. In our intravital experiments, the exteriorization of the subiliac LN likely induces P-selectin expression on the surface of the endothelial cells. Peyer's patch HEVs express P-selectin under intravital microscopy (38). We observed that an LPS-stimulated LN showed a strong staining for P-selectin in HEVs. P-selectin is also reported to be expressed on the HEVs of inflamed LNs during infection in a model of experimental murine listeriosis (41). Thus, although the interaction of PSGL-1 with P-selectin mediates only 10% of leukocyte rolling in our experimental settings, it is likely that this interaction contributes more to leukocyte rolling during inflammation and infection. In addition, E-selectin is expressed on the HEVs of inflamed PLNs and is involved in the recruitment of plasmacytoid dendritic cells into inflamed PLNs (42, 43). Since PSGL-1 functions not only as a P-selectin ligand but also as an E-selectin ligand, PSGL-1-mediated leukocyte rolling may also play a role in leukocyte trafficking to the PLNs when E-selectin is expressed on the HEVs.

The physiological significance of PSGL-1-dependent leukocyte rolling in the HEVs of PLNs remains to be determined. Given that intravital microscopy of the subiliac LN requires exteriorization of the LN, which probably causes inflammation, the observed leukocyte interaction with the vessel wall probably does not simply reflect leukocyte behavior during homeostasis, but rather their behavior under inflammatory conditions. Since P-selectin is expressed in response to inflammatory stimuli, the PSGL-1 interaction with P-selectin may play a role in the entry of cells, such as P-selectin-binding memory T cells, into the inflamed LN. In this regard, the administration of activated platelets has been shown to induce lymphocyte rolling in HEVs in the absence of functional L-selectin (11). This induction of lymphocyte rolling is currently thought to be mediated by P-selectin on circulating activated platelets, which supports simultaneous platelet adhesion to PNAd on HEVs and to PSGL-1 on lymphocytes. This L-selectin-independent mechanism of lymphocyte adhesion to HEVs restores lymphocyte trafficking to the PLNs, and reconstitutes T cell-mediated immunity in response to a cutaneous antigen (12). Similarly, it is possible that P-selectin expressed on HEVs in response to inflammatory stimuli directly mediates memory cell rolling in HEVs via interaction with PSGL-1, leading to memory lymphocyte trafficking to the PLNs. Among memory T cells, P-selectin-binding cells are observed both in L-selectinhigh and L-selectinlow populations, which correspond to central memory and effector memory populations, respectively. Central memory T cells migrate to LNs, and upon a secondary challenge with antigen, can efficiently stimulate dendritic cells, help B cells and generate new wave of effector cells (44). It is interesting to speculate that the PSGL-1-mediated interaction contributes to more efficient trafficking of central memory T cells to the inflamed LNs draining the site of antigen challenge or infection. It is also possible that PSGL-1 mediates the entry of effector memory T cells directly via HEVs, leading to rapid effector functions in situ in the inflamed LNs. Another possibility is that PSGL-1-mediated rolling on P-selectin expressed on inflamed HEVs may mediate a secondary tethering of leukocytes, thereby capturing flowing L-selectin-expressing cells, such as naive lymphocytes, through PSGL-1–L-selectin interactions. These interactions may facilitate direct interactions between the tethered cells and PNAd expressed on the endothelial surface, thus amplifying the entry of naive lymphocytes into the inflamed LN.

Lymphocyte migration into the PLNs is a critical component of the immune response. In the current study, we have shown that PSGL-1 interacting with P-selectin mediates L-selectin-independent leukocyte rolling in the HEVs of PLNs. Because P-selectin is expressed in a variety of inflammatory conditions, this PSGL-1-mediated leukocyte rolling in HEVs may play a role in leukocyte trafficking to PLNs under various inflammatory conditions.


   Acknowledgements
 
This work was supported by a Grant-in-Aid for the 21st Century Center of Excellence Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Japan.


   Abbreviations
 
APC, allophycocyanin
HEV, high endothelial venule
LFA-1, leukocyte function-associated antigen
LN, lymph node
PLN, peripheral lymph node
PNAd, peripheral node addressin
PSGL-1, P-selectin glycoprotein ligand-1

   Notes
 
Transmitting editor: H. Karasuyama

Received 27 September 2006, accepted 22 December 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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