Efficient Lymphocyte Migration Across High Endothelial Venules of Mouse Peyer’s Patches Requires Overlapping Expression of L-Selectin and β₇ Integrin¹

The continuous migration of lymphocytes to peripheral and mucosal lymphoid tissues is largely mediated by lymphocyte cell surface receptors binding to their ligands expressed on specialized postcapillary venules termed high endothelial venules (HEV)³ (1, 2, 3, 4). Lymphocyte/HEV interactions in peripheral lymph nodes (PLNs) are predominantly regulated by L-selectin (CD62L), the constitutively expressed leukocyte adhesion molecule identified by the MEL-14 mAb in mice (5, 6). Early studies suggested that L-selectin primarily mediates lymphocyte attachment to HEV of PLNs and mesenteric lymph nodes (MLNs), while migration to Peyer’s patch HEV is primarily controlled by the integrin α₄β₇ (5, 7, 8, 9, 10, 11). Lymphocyte attachment to Peyer’s patch HEV during in vitro assays is specifically and completely blocked by Abs against α₄ or β₇ integrin chains (12). However, lymphocyte migration into mouse Peyer’s patches is partially inhibited by Fab of the MEL-14 mAb (13), and mouse Peyer’s patch HEV express low but functional levels of L-selectin carbohydrate ligands (14). The generation of L-selectin-deficient (L-selectin^−/−) mice confirmed that L-selectin significantly influences lymphocyte migration into Peyer’s patches and MLNs (6). This has been further supported by intravital microscopic analysis of isolated Peyer’s patches demonstrating that both L-selectin, and to a lesser degree, α₄β₇ integrins participate in the initial interaction between lymphocytes and HEV as well as in subsequent rolling (15, 16). Consistent with this, α₄β₇ and α₄β₁ integrins can each mediate rolling and firm adhesion of lymphocytes during in vitro assays (17, 18, 19).

L-selectin^−/− mice have a significant reduction (70–90%) in the number of resident lymphocytes within PLNs, but are viable, have no developmental defects, and do not succumb to multifocal infections (6, 20, 21). Lymphocytes from L-selectin^−/− mice are completely deficient in their ability to attach to PLN HEV during in vitro binding assays and are unable to migrate across HEV of resting or responsive PLNs in normal mice during short-term (1 h) or long-term (48 h) in vivo migration experiments (6, 20, 21, 22). Migration of L-selectin^−/− lymphocytes into Peyer’s patches and MLNs is also dramatically reduced in short-term migration assays, but this deficit largely recovers during long-term assays (6, 22). The ability of L-selectin^−/− lymphocytes to reconstitute Peyer’s patches to near wild-type levels, and MLNs to a lesser extent, in long-term migration assays suggests that either α₄β₇ mediates a low level of L-selectin-independent cell capture/entry from the blood or alternatively that adhesion molecules other than α₄β₇ substitute for L-selectin in these tissues. L-selectin^−/− mice also demonstrate decreased leukocyte rolling at sites of inflammation, decreased leukocyte recruitment into an inflamed peritoneum, decreased delayed-type hypersensitivity responses, delayed rejection of allogeneic skin transplants, and resistance to LPS-induced septic shock, but have augmented humoral and cellular immune responses (6, 23, 24, 25, 26, 27).

Lymphocyte migration into lymphoid tissues is also influenced by the β₇ integrins: the β₇ chain associates with two α-chains to generate α₄β₇ and α_Eβ₇ (7, 12, 28, 29). α_Eβ₇ is primarily expressed on intraepithelial lymphocytes and lymphocytes migrating to the mucosa (30, 31, 32, 33). By contrast, the majority of mouse lymphocytes express α₄β₇, which interacts with the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) to mediate Peyer’s patch and MLN migration (27, 34, 35, 36). MAdCAM-1 is expressed on HEV located within Peyer’s patches and MLN, on vessels within the lamina propria, and on follicular dendritic cells within Peyer’s patches and chronically inflamed PLN and spleen (37, 38, 39). β₇ integrin-deficient (β₇ integrin^−/−) mice are healthy, viable, and develop normally (16). These mice have dramatically hypocellular Peyer’s patches that contain only rudimentary follicles, and have lamina propria and intraepithelial lymphocyte numbers that are significantly reduced. Migration of β₇ integrin^−/− lymphocytes into Peyer’s patches is severely reduced during short-term in vivo assays, while lymphocyte migration into MLNs is diminished and migration into PLNs is normal (16). Studies using function-blocking mAbs have demonstrated that L-selectin and α₄β₇ initiate lymphocyte interactions with Peyer’s patch HEV, and L-selectin and α₄β₇ both participate in rolling during acute migration assays in situ (15). Furthermore, α₄β₇ dramatically reduces lymphocyte rolling velocities, an apparent requirement for LFA-1 engagement. In additional studies, L-selectin was necessary for 80–90% of lymphocyte rolling in Peyer’s patch HEVs of wild-type and β₇ integrin^−/− mice, while β₇ integrins were required for adhesion and emigration during acute migration assays in situ (16, 40). Furthermore, leukocyte rolling on Peyer’s patch HEV occurs at different characteristic velocities with slow rolling predominating in L-selectin^−/− mice and fast rolling characterizing β₇ integrin^−/− mice (40). Therefore, both L-selectin and the β₇ integrins serve major roles in lymphocyte migration to gut-associated lymphoid tissues.

A dual migratory specificity for Peyer’s patches and MLNs involving L-selectin and α₄β₇ integrins establishes a framework for understanding lymphocyte migration into these tissues. Because most circulating lymphocytes express both L-selectin and β₇ integrins (27, 41), α₄β₇ could mediate selectin-independent cell capture from the blood, thus explaining the ability of L-selectin^−/− lymphocytes to enter Peyer’s patches and MLNs in long-term migration assays. However, it is relevant that significantly more L-selectin^−/− lymphocytes enter the spleen than wild-type lymphocytes during migration assays and that L-selectin^−/− mice have 30–50% larger spleens than their wild-type littermates. By contrast, Peyer’s patches and MLNs are not increased in size in L-selectin^−/− mice (6, 22). Thus, it remains unresolved as to whether these two molecules account for all lymphocyte migration into Peyer’s patches and MLNs or whether these two receptors have additive functions. Therefore, mice deficient in both L-selectin and β₇ integrins (L-selectin/β₇ integrin^−/−) were generated to further characterize interactions between these adhesion molecules during lymphocyte recirculation. Lymphocyte migration into Peyer’s patches was examined in these studies because the lack of afferent lymphatics precludes lymphocyte entry though other routes (42), which thereby provides an excellent tissue for examining lymphocyte/HEV interactions. The phenotype of L-selectin/β₇ integrin^−/− mice demonstrated a requirement for both L-selectin and β₇ integrin expression for optimal lymphocyte migration into Peyer’s patches with lymphocyte migration into this tissue being eliminated in L-selectin/β₇ integrin^−/− mice.

L-selectin^−/− mice were generated as described (6) and backcrossed onto the C57BL/6 background for 7–10 generations. β₇ integrin^−/− mice were generated as described (16). Mice lacking both L-selectin and β₇ integrins were generated by crossing F₁ offspring from crosses of homozygous L-selectin^−/− mice with homozygous β₇ integrin^−/− mice as described (43). Cell surface L-selectin or β₇ integrin expression by lymphocytes was assessed by two-color fluorescence cytometry. All mice used were 2–3 mo of age and were housed in a specific pathogen-free barrier facility. Control mice were age-matched wild-type mice generated from heterozygous breedings of L-selectin^+/−/β₇ integrin^+/− mice or were C57BL/6 mice purchased from The Jackson Laboratory (Bar Harbor, ME). Equivalent results were obtained for both groups of control mice, and both groups are referred to subsequently as wild-type mice. All studies and procedures were approved by the Animal Care and Use Committee of Duke University.

Blood was aspirated from the retroorbital venous plexus of anesthetized mice. Single-cell suspensions from spleen, PLN (bilateral axillary and inguinal nodes were pooled), MLN (superior mesenteric cords were pooled), and Peyer’s patches were prepared as described (22) and filtered through nylon gauze to remove debris before washing twice in PBS. Erythrocytes in splenocyte suspensions were lysed with Tris-buffered 0.1 M ammonium chloride solution before labeling. Total cell numbers were enumerated using a hemocytometer. For counting blood leukocytes, erythrocytes were lysed with 2% acetic acid.

Abs used in these studies included unconjugated, FITC-, or biotin-conjugated mAbs reactive with L-selectin (LAM 1–110, 44 , β₇ integrin (M293; PharMingen, San Diego, CA), and α₄β₇ integrin (DATK-32; American Type Culture Collection, Manassas, VA). Phycoerythrin (PE)-conjugated streptavidin was used to reveal biotinylated Ab staining and PE-conjugated goat anti-rat IgG Abs (both from Southern Biotechnology, Birmingham, AL) were used to detect α₄β₇ integrin labeling. Isotype-matched rat IgG Abs (PharMingen) were used as controls. Following staining, blood erythrocytes were lysed with Coulter whole-blood Immuno-Lyse kit according to the manufacturer’s instructions (Coulter, Miami, FL). Ten to 30,000 cells with the forward and side light scatter properties of lymphocytes were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) with fluorescence intensity shown on a 4-decade log scale. Fluorescence contours are shown as 50% log density plots.

Lymphocyte binding to PLN, MLN, and Peyer’s patch HEV was performed as previously described (22, 45). Briefly, splenocytes were isolated from wild-type, L-selectin^−/−, β₇ integrin^−/−, and L-selectin/β₇ integrin^−/− mice. Following lysis of erythrocytes, cells were washed three times in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 5% FCS and 10 mM HEPES, adjusted to 25 × 10⁶ cells/ml, and kept on ice until used. Then, 200 μl of the above cell suspension was overlayered onto 12-μm thick frozen sections of PLN, MLN, and Peyer’s patches from wild-type mice. The slides were rotated at 64 rpm for 30 min at 4°C. Cells were then gently dumped off the slides and the slides were placed vertically into 2.4% buffered glutaraldehyde overnight at 4°C. The slides were washed in PBS, counterstained in hematoxylin, and mounted under glass coverslips. For each experiment, splenocytes from each of the four groups of mice were assayed for binding to at least two sections from each tissue.

Two-color lymphocyte migration experiments were as described (6, 22). Briefly, single-cell suspensions were prepared from the spleens of donor mice and labeled with calcein-AM (Molecular Probes, Eugene, OR). Splenocytes (5–10 × 10⁷) were incubated in 2 ml of RPMI 1640 medium containing 1 μM calcein on ice for 30 min with gentle mixing every 5 min. Cells were then washed twice in PBS and counted. Internal control wild-type splenocytes were labeled with PKH26 (Sigma, St. Louis, MO). Splenocytes (5 × 10⁷) were resuspended in 1 ml of PKH26 diluent, immediately added to 1 ml of a 3 μM PKH26 dye solution, and incubated for 2 min at room temperature. For some experiments, β₇ integrin^−/− splenocytes were used as an internal control. Labeling was stopped by the addition of 2 ml of FCS. Cell suspensions were washed twice with PBS and counted. Equal numbers (2 × 10⁷) of calcein-labeled and PKH26-labeled cells were mixed, pelleted, and resuspended in a total volume of 400 μl of PBS for injection into the lateral tail veins of individual wild-type mice. An aliquot of the injected cell mixture was analyzed by flow cytometry to calculate the injected ratio of calcein- to PKH26-labeled cells (R_i). After either 1 or 48 h of migration, single-cell suspensions of tissues and blood were prepared, and the percentage of calcein- and PKH26-labeled cells was determined by flow cytometric analysis. Two to 5000 PKH26-labeled cells were collected for each sample. The ratio of calcein-/PKH26-labeled cells within tissues or blood (R_o) was calculated, and results were expressed as the ratio of R_o/R_i in each tissue, as described (6, 22). The total number of calcein- or PKH26-labeled cells recovered from individual lymphoid tissues or blood was determined by multiplying the total cell counts for individual tissues by the frequency of labeled cells.

All data are shown as mean values ± SEM. Comparisons between groups were conducted using the Student’s t test.

L-selectin^−/− and β₇ integrin^−/− mice were crossed to generate mice homozygous deficient at both loci (L-selectin/β₇ integrin^−/−) as described (43). Each group of mice was free from obvious signs of pathology or disease for up to 1 yr of age. Leukocytes from L-selectin/β₇ integrin^−/− mice completely lacked cell surface expression of both L-selectin and β₇ integrins (Fig. 1, A and B). Both circulating and splenic leukocytes from L-selectin^−/− mice expressed wild-type levels of β₇ integrins, while leukocytes from β₇ integrin^−/− mice expressed wild-type levels of L-selectin (Table I). Significant differences in the percentage of circulating or splenic leukocytes expressing either L-selectin or β₇ integrins were not observed in β₇ integrin^−/− or L-selectin^−/− mice, respectively, compared with wild-type mice (Table I). Thus, the loss of one adhesion molecule did not lead to significant changes in expression intensity or frequency of the other adhesion molecule. However, circulating leukocytes expressing both L-selectin and β₇ integrins were present at a higher frequency compared with spleen cells in wild-type mice as previously reported (27).

L-selectin and β₇ integrin loss was confirmed by determining the ability of splenocytes from L-selectin/β₇ integrin^−/− mice to bind to HEV of Peyer’s patch, MLN, and PLN during in vitro frozen-section binding assays (45). The loss of both L-selectin and β₇ integrin expression resulted in a ≥95% reduction in lymphocyte binding to HEV of each lymphoid tissue (Fig. 1,C). L-selectin loss resulted in an ∼30% decrease in splenocyte binding to HEV of Peyer’s patch, a 75% decrease in binding to MLN HEVs, and a 98% decrease in binding to PLN HEVs. Loss of β₇ integrin expression resulted in a 97% and 46% reduction in splenocyte binding to Peyer’s patch and MLN HEVs, respectively, without a significant loss in binding to PLN HEVs (Fig. 1 C). Therefore, L-selectin and β₇ integrins collectively mediate the vast majority of lymphocyte binding to HEV under these in vitro conditions.

The loss of both L-selectin and β₇ integrins severely affected the distribution of lymphocytes within Peyer’s patches from 2-mo-old L-selectin/β₇ integrin^−/− mice. Most Peyer’s patches from L-selectin/β₇ integrin^−/− mice were virtually undetectable macroscopically with an average of 3.5 ± 0.4 (range 2–5; p < 0.001 vs wild-type; n = 12) Peyer’s patches being identified in each mouse. The most easily identified Peyer’s patches showed no protrusion from the gut wall and were nearly translucent in color. This was consistent with a significant reduction in numbers of lymphocytes present in histologic sections of these Peyer’s patches (Fig. 2). However, despite the small size and significant reduction in cellularity of Peyer’s patches from L-selectin/β₇ integrin^−/− mice, rudimentary follicles were still observed (Fig. 2). Microscopic examination of serial sections across the entire gut revealed an equal number of Peyer’s patches in both wild-type and L-selectin/β₇ integrin^−/− mice, as described for β₇ integrin^−/− mice (16). Macroscopic examination revealed equal numbers (range of 6–12) of readily detectable Peyer’s patches that were microscopically similar protruding from the gut wall in both wild-type and L-selectin^−/− mice (Fig. 2). Normal numbers of Peyer’s patches were visually identified and harvested from β₇ integrin^−/− mice (7.7 ± 0.6 per mouse, range 6–10, n = 6). However, Peyer’s patches of β₇ integrin^−/− mice were markedly reduced in size and protruded only minimally from the gut wall, making them macroscopically less apparent (Fig. 2, and 16 . Thus, the combined loss of L-selectin and β₇ integrin expression had a major effect on Peyer’s patch cellularity, but not its overall gross architecture.

The numbers of lymphocytes within Peyer’s patches of L-selectin/β₇ integrin^−/− mice were reduced significantly beyond the decreases resulting from the loss of either L-selectin or β₇ integrin expression alone. Specifically, Peyer’s patches of L-selectin/β₇ integrin^−/− mice were reduced in cellularity by >99% in comparison with wild-type mice (Table II). This may be a slight underestimate of the effect of L-selectin/β₇ integrin loss because the small size of Peyer’s patches from these mice resulted in a higher recovery of intraepithelial lymphocytes that surrounded the Peyer’s patches and contaminated the Peyer’s patch cell populations to varying degrees (data not shown). However, as stated above, the smallest of the Peyer’s patches from the L-selectin/β₇ integrin^−/− mice were not identified macroscopically and thus were not harvested or included in the cell counts. Peyer’s patches in β₇ integrin^−/− mice were reduced by ∼90% as previously described (16), which was significantly different from wild-type or L-selectin^−/− mice. These results indicate that L-selectin and β₇ integrins account for the vast majority of lymphocyte migration into Peyer’s patches.

Lymphocyte numbers within MLNs of L-selectin/β₇ integrin^−/− mice were reduced by 92% relative to wild-type mice (Table II, and 43 . L-selectin^−/− mice had an ∼45% reduction, while β₇ integrin^−/− and wild-type mice had similar numbers of MLN lymphocytes. By contrast, the loss of L-selectin expression resulted in a 95% decrease in PLN lymphocytes, but the number of resident lymphocytes within PLNs of L-selectin^−/− mice tended to be increased with the additional loss of β₇ integrin expression (Table II). β₇ integrin loss also resulted in a 10% increase in PLN cellularity. These results are consistent with overlapping functions for L-selectin and β₇ integrins during lymphocyte entry into MLNs, but point out that β₇ integrin expression does not contribute substantially to lymphocyte migration into PLNs.

Circulating leukocyte numbers were markedly increased in L-selectin/β₇ integrin^−/− mice relative to wild-type mice (by 260%, Table II). By contrast, circulating leukocyte numbers were not significantly different between wild-type and L-selectin^−/− mice and were only slightly elevated in β₇ integrin^−/− mice. Increased lymphocyte numbers accounted for the vast majority of the increase in circulating leukocytes in L-selectin/β₇ integrin^−/− mice. Smaller, yet significant, relative increases in numbers of circulating neutrophils, monocytes, and eosinophils were also observed in these mice compared with wild-type controls. L-selectin^−/− mice had significantly increased numbers of circulating monocytes (∼130% increase) compared with wild-type controls as previously described (22). Mice deficient in β₇ integrins alone had slightly increased numbers of circulating neutrophils, lymphocytes, and eosinophils relative to wild-type mice (Table II).

The loss of L-selectin resulted in an ∼60% increase in spleen lymphocytes, which was further increased by 49% with the additional loss of β₇ integrin expression (Table II). There was a trend toward increased spleen cellularity with the loss of β₇ integrin expression, although this was a variable finding. Therefore, the combined loss of L-selectin and the β₇ integrins had an additive effect on the number of circulating leukocytes and the number of lymphocytes that accumulated within the spleen.

The extent that L-selectin and the β₇ integrins regulate lymphocyte entry into lymphoid tissues was assessed in short-term (1 h) in vivo migration assays. Splenocytes from wild-type, L-selectin^−/−, β₇ integrin^−/−, and L-selectin/β₇ integrin^−/− mice were labeled with calcein, mixed with an equal number of PKH26-labeled wild-type splenocytes (internal control), and injected i.v. into wild-type mice. The frequency of labeled lymphocytes within tissues was determined after 1 h. Differences in migration patterns were assessed by comparing the ratio of calcein-labeled test cells to PKH26-labeled internal control cells present within each tissue (R_o) with the ratio of calcein-labeled test cells to PKH26-labeled control cells that were injected (R_i). Splenocytes were used for these studies because they represent mixed populations of T and B cells, contain both naive and memory cell populations, and have the most heterogeneous distribution of L-selectin and β₇ integrin expression (27). Nonetheless, the distribution of lymphocyte populations present within the spleens of 2-mo-old L-selectin/β₇ integrin^−/− mice was not dramatically different from that of wild-type mice (D.A.S., X.-Q.Z., N.W., and T.F.T., manuscript in preparation), β₇ integrin^−/− mice (16) or L-selectin^−/− mice (22).

The migration of L-selectin/β₇ integrin^−/− lymphocytes into Peyer’s patches was dramatically decreased compared with wild-type (99% decrease, p < 0.001), L-selectin^−/− (96% decrease, p < 0.001), or β₇ integrin^−/− lymphocytes (85% decrease, p < 0.01) (Fig. 3). Similarly, the migration of double-deficient lymphocytes into MLNs was significantly decreased compared with wild-type (by 99%, p < 0.001), L-selectin^−/− (by 77%, p < 0.01), or β₇ integrin^−/− (by 98%, p < 0.001) lymphocytes (43). The combined loss of L-selectin and β₇ integrin expression resulted in a reduction of lymphocyte migration to PLNs similar to the decrease observed for L-selectin^−/− lymphocytes (Fig. 3). The increase in L-selectin/β₇ integrin^−/− lymphocyte migration to the spleen was also similar to that seen for L-selectin^−/− lymphocytes (Fig. 3). The combined loss of both L-selectin and β₇ integrin expression increased the number of lymphocytes remaining in the blood after 1 h by 25% (p < 0.01) compared with wild-type lymphocytes. Therefore, the L-selectin/β₇ integrin^−/− deficiency significantly reduced lymphocyte migration into Peyer’s patches and MLNs beyond the level seen in the single-deficient lymphocytes, while enhancing blood and spleen localization.

To more precisely quantify and verify that L-selectin/β₇ integrin^−/− lymphocyte migration was different from that of β₇ integrin^−/− lymphocytes, splenocytes from L-selectin/β₇ integrin^−/− mice were labeled with calcein and mixed with an equal number of internal control PKH26-labeled β₇ integrin^−/− splenocytes. One hour after i.v. injection into wild-type mice, there was a 96% reduction in migration of L-selectin/β₇ integrin^−/− lymphocytes into Peyer’s patches relative to the migration of β₇ integrin^−/− lymphocytes (p < 0.01, Fig. 4). There was also a 97% decrease in L-selectin/β₇ integrin^−/− lymphocyte migration into MLNs and a 98% reduction of migration into PLNs as compared with β₇ integrin^−/− lymphocytes (p < 0.01). Therefore, L-selectin is necessary for the efficient migration of wild-type and β₇ integrin^−/− lymphocytes into Peyer’s patches and MLN and the combined effect of these deficiencies is more than additive.

To assess how L-selectin and β₇ integrin loss affected the accumulation of recirculating lymphocytes within tissues after the cells had the opportunity to reach equilibrium, in vivo migration assays were conducted as described above except lymphocyte localization within tissues was assessed at 48 h. Lymphocytes deficient in both L-selectin and β₇ integrin expression were excluded from entering Peyer’s patches (>99% decrease, p < 0.001) relative to wild-type lymphocytes (Fig. 5). Furthermore, lymphocyte localization within MLNs (>99% decrease, p < 0.001) and PLNs (98% decrease, p < 0.001) was also significantly affected when compared with wild-type lymphocytes (Fig. 5). By contrast, β₇ integrin^−/− lymphocytes showed a 94% decrease in Peyer’s patch migration and a modest 34% decrease in MLN migration when compared with wild-type splenocytes (Fig. 5). The number of circulating L-selectin/β₇ integrin^−/− lymphocytes remained increased at 48 h (by ∼60%, p < 0.01) relative to splenocytes from all other groups of mice (Fig. 5). The migration of L-selectin/β₇ integrin^−/− lymphocytes into the spleen was increased by twofold relative to wild-type lymphocytes (p < 0.001) and tended to be increased slightly relative to L-selectin^−/− lymphocytes (Fig. 5). These results show that the L-selectin/β₇ integrin deficiency eliminated the vast majority of lymphocyte recirculation through Peyer’s patches, MLNs, and PLNs.

Conclusions similar to those discussed above were obtained when the numbers of splenocytes that migrated into tissues at 1 and 48 h were determined (Table III). Within Peyer’s patches, L-selectin/β₇ integrin^−/− lymphocyte migration was <1% of wild-type levels at 1 h and ∼0.2% at 48 h. Within MLNs, L-selectin/β₇ integrin^−/− lymphocyte migration was <2% of wild-type levels at 1 h and <1% at 48 h. Similarly, L-selectin/β₇ integrin^−/− lymphocyte migration into PLNs was ∼6% of wild-type levels at 1 h and ∼1.5% at 48 h. These results indicate that the L-selectin/β₇ integrin deficiency affects the majority of splenocyte migration including both naive and memory subpopulations. Two additional observations were of interest. First, despite the L-selectin/β₇ integrin deficiency a very small number of lymphocytes were nonetheless able to enter Peyer’s patches and MLNs at 1 h (Table III). These emigrant cells were lymphocytes, although their identity or whether they represented lymphocytes present within the vasculature of the harvested tissues was not assessed. However, the number of labeled L-selectin/β₇ integrin^−/− lymphocytes within Peyer’s patches and MLNs did not increase over the subsequent 48 h. This is in stark contrast to results obtained using splenocytes of wild-type or single-deficient mice. Specifically, migration of wild-type lymphocytes to Peyer’s patches and MLNs at 48 h increased by four- and threefold, respectively, relative to 1 h numbers (p < 0.001, Table III). Similar increases in numbers of L-selectin^−/− lymphocytes migrating to Peyer’s patches and MLNs (6.3- and 3.7-fold, respectively, p < 0.05) were observed over this time period. β₇ integrin^−/− lymphocytes showed significant but smaller increases in numbers of migrating lymphocytes over the 48-h time period (∼2.3-fold, p < 0.02) relative to wild-type or L-selectin^−/− cells. Therefore, in contrast to L-selectin/β₇ integrin^−/− lymphocytes, these results indicate that wild-type and single-deficient lymphocytes accumulate within these tissues.

A second interesting result was that the number of L-selectin/β₇ integrin^−/− lymphocytes entering PLNs was two- to threefold greater than the number of L-selectin^−/− lymphocytes at both 1 and 48 h (Table III). This observation appears valid because the number of resident lymphocytes within PLNs of L-selectin/β₇ integrin^−/− mice tended to be greater than in L-selectin^−/− littermates (Table II). Although the numbers of L-selectin^−/− or L-selectin/β₇ integrin^−/− lymphocytes migrating into PLNs did not increase from 1 to 48 h, numbers of wild-type or β₇ integrin^−/− cells increased ∼3.5-fold (Table III, p < 0.01). Within the circulation, the number of wild-type or single-deficient lymphocytes decreased by 40–50% over the 48-h time period (Table III). By contrast, the number of circulating L-selectin/β₇ integrin^−/− lymphocytes only decreased a modest 20% from 1 to 48 h. These results further support the observations that lymphocytes lacking L-selectin and β₇ integrin expression are unable to leave the circulation and enter lymphoid tissues such as Peyer’s patches, PLNs, and MLNs and are consistent with the significantly elevated numbers of circulating lymphocytes observed in L-selectin/β₇ integrin^−/− mice (Table II).

Whether the loss of L-selectin or β₇ integrin expression skewed Peyer’s patch lymphocyte expression of β₇ integrins or L-selectin, respectively, was assessed in adhesion molecule-deficient mice. As observed for circulating lymphocytes (Fig. 1,A), L-selectin loss did not affect the level of β₇ integrin expression by lymphocytes within Peyer’s patches (Fig. 6). Most lymphocytes within Peyer’s patches of wild-type (94 ± 1% positive, n = 4) and L-selectin^−/− (92 ± 1%, n = 4) mice expressed α₄β₇ at comparable intensities. By contrast, the frequency of L-selectin expressing lymphocytes in β₇ integrin^−/− mice (45 ± 5%, n = 4) was significantly lower than in wild-type mice (75 ± 4%, n = 3, p < 0.002), although L-selectin was expressed at similar intensities by most L-selectin-bearing lymphocytes (Fig. 6). The lower frequency of L-selectin expressing lymphocytes in Peyer’s patches of β₇ integrin^−/− mice compared with blood lymphocytes (Fig. 1) is likely to reflect the slower entry of circulating lymphocytes into this tissue in the absence of β₇ integrin expression. As a result, the majority of Peyer’s patch lymphocytes in β₇ integrin^−/− mice are likely to have been long-term residents within this tissue or may have arisen from in situ proliferation. This alters the lymphocyte subset distribution within Peyer’s patches of β₇ integrin^−/− mice compared with wild-type mice, but these changes are relatively minor (D.A.S., X.-Q.Z., N.W., and T.F.T., manuscript in preparation). An additional previously discussed influence on L-selectin expression is that Peyer’s patch preparations from L-selectin/β₇ integrin^−/− mice contain some degree of contaminating gut lymphocytes that typically do not express L-selectin.

In this study, L-selectin and the β₇ integrins regulated the vast majority of lymphocyte migration across HEVs of Peyer’s patches, MLNs, and other peripheral lymphoid tissues. The simultaneous genetic loss of both L-selectin and β₇ integrin expression prevented the majority of lymphocytes (>95% inhibition) from attaching to HEV of Peyer’s patches during in vitro binding assays (Fig. 1). Moreover, the inability to bind HEV eliminated the vast majority of L-selectin/β₇ integrin^−/− lymphocyte migration into Peyer’s patches during short-term in vivo migration assays (>99% inhibition, p < 0.01, Fig. 3, Table III) as well as the long-term localization of lymphocytes in these tissues (>99% inhibition, p < 0.01, Fig. 5, Table III). The lack of lymphocyte migration into Peyer’s patches correlated directly with the dramatically reduced size (Fig. 2) and cellularity (99% reduced) of this tissue in L-selectin/β₇ integrin^−/− mice (Table II). In addition, the increased numbers of injected L-selectin/β₇ integrin^−/− lymphocytes remaining in the blood of wild-type mice after 48 h (Fig. 5) correlated with the markedly increased numbers of circulating lymphocytes found in L-selectin/β₇ integrin^−/− mice (Table II). While loss of either L-selectin or, to a greater extent, the β₇ integrins alone resulted in significant inhibition of Peyer’s patch migration, neither produced complete inhibition. Collectively, these results indicate that L-selectin and β₇ integrins primarily interact along the same adhesion pathway to mediate the vast majority of lymphocyte migration into Peyer’s patches, although each molecule can function independent of the other, albeit less efficiently.

The current study of lymphocyte migration into Peyer’s patches extends prior in situ studies (15, 16, 40) by demonstrating that the combined absence of L-selectin and β₇ integrins eliminates the vast majority of lymphocyte migration into mouse Peyer’s patches (Figs. 3 and 5). Because the current studies circumvent the inflammatory influences introduced by the invasive surgical procedures necessary to view leukocyte/endothelial interactions in situ (15, 16, 40), it is likely that L-selectin and the β₇ integrins are the essential receptors for normal migration during noninflammatory situations. The absence of lymphocyte migration into Peyer’s patches even after 48 h suggests that other adhesive mechanisms are unlikely to compensate significantly for the loss of both L-selectin and β₇ integrins during migration into reactive Peyer’s patches because other adhesion molecules are unable to compensate for L-selectin loss during lymphocyte entry into reactive PLNs across HEVs (22). Therefore, a remaining issue is the identity of the adhesion molecules that facilitate lymphocyte migration into Peyer’s patches in the absence of L-selectin or β₇ integrin expression. P-selectin has recently been shown to support a low level of leukocyte rolling along Peyer’s patch venules from L-selectin/β₇ integrin^−/− mice following exteriorization (40). It has also been postulated that P-selectin facilitates the entry of lymphocytes into lymphoid tissues across HEVs by the formation of lymphocyte-platelet aggregates (46, 47). However, because lymphocytes do not migrate across HEVs of resting or activated PLNs in the absence of L-selectin expression (22) and circulating lymphocyte-platelet aggregates are not encountered under normal physiologic conditions, this mechanism may only contribute modestly to lymphocyte entry into lymphoid tissues. Therefore, it is likely that α₄β₇ integrins mediate lymphocyte capture and rolling on Peyer’s patch HEV in the absence of L-selectin expression, albeit at much lower levels of efficiency than the combination of L-selectin and β₇ integrins. Previous studies have documented a role for LFA-1 during lymphocyte migration into lymphoid tissues (48), and L-selectin and ICAM-1 function cooperatively to mediate leukocyte rolling at sites of inflammation (26). Consistent with this, migration of β₇ integrin^−/− lymphocytes into Peyer’s patches is further significantly reduced with the additional loss of ICAM-1 expression (D.A.S., X.-Q.Z., and T.F.T., unpublished observations). Therefore, it is likely that these molecules retard lymphocyte rolling velocities sufficiently to permit the formation of firm adhesive interactions that proceed lymphocyte migration into Peyer’s patches in the absence of β₇ integrin expression. However, in the absence of L-selectin and β₇ integrin expression, these alternative adhesive mechanisms are inadequate to mediate lymphocyte migration into Peyer’s patches.

T cells migrate into Peyer’s patches and all other lymphoid tissues at a faster tempo than B cells, in part, due to their expression of 50–100% higher levels of L-selectin compared with B cells (27). Although B cells express α₄β₇ integrins at twofold higher levels than T cells, more T cells still migrate into Peyer’s patches. In the absence of L-selectin expression, the vast majority of PLN lymphocytes (which are predominantly T cells) are unable to enter Peyer’s patches (22) despite the fact that the majority of these cells express α₄β₇ integrins (27). In fact, even in the absence of L-selectin expression, spleen T cells migrate into Peyer’s patches at a fivefold higher rate than spleen B cells (27). However, β₇ integrin loss appeared to have a more significant effect on splenocyte/Peyer’s patch HEV interactions than the loss of L-selectin in the current studies (Figs. 3 and 5). This apparently contradictory finding points out a need for further examination of the differential roles for L-selectin and β₇ integrins in short-term T and B cell subset migration. For example, a loss of β₇ integrin expression by B cells in combination with their lower levels of L-selectin expression may significantly diminish or preclude their entry into Peyer’s patches. Although many of the effects of adhesion receptor loss described in the current study were quite large and thereby were likely to involve most or all lymphocyte subsets, heterogeneity of L-selectin and β₇ integrin expression on some minor lymphocyte subsets may be of substantial importance in directing their trafficking to selective tissues (49). Nonetheless, the results discussed above demonstrate that adhesion molecule requirements during migration may not be generally equivalent between lymphocytes isolated from different lymphoid tissues.

In addition to facilitating lymphocyte/HEV interactions, we have postulated previously that the β₇ integrins retain lymphocytes within Peyer’s patches subsequent to their entry across HEV, particularly B cells (27). The current studies validate this hypothesis. The entry of L-selectin^−/− and β₇ integrin^−/− lymphocytes into Peyer’s patches at 1 h is inhibited by 48–70% and 89% relative to lymphocytes from wild-type mice, respectively (Table III and 27 . However, by 48 h the number of emigrant L-selectin^−/− lymphocytes within Peyer’s patches is similar to that of wild-type lymphocytes (Table III). Peyer’s patches in L-selectin^−/− and wild-type mice are also similar in cellularity (6, 22). Therefore, although the loss of L-selectin greatly reduces the tempo of lymphocyte migration into Peyer’s patches, emigrant L-selectin^−/− lymphocytes can populate this tissue to near wild-type levels by 48 h. By contrast, the number of newly emigrated β₇ integrin^−/− lymphocytes within Peyer’s patches by 48 h is 93% below that of wild-type lymphocytes (Table III). Similarly, the cellularity of Peyer’s patches in β₇ integrin^−/− mice is reduced by 90% (Table II and 16 . Therefore, the bulk of lymphocytes that enter Peyer’s patches are not retained in the absence of β₇ integrin expression. This is perhaps consistent with the recent observation that MAdCAM-1 is expressed at sites within lymphoid tissues in addition to its display by HEV (37).

L-selectin and α₄β₇ have been predominantly hypothesized to direct the selective migration of lymphocytes to peripheral lymph nodes and the gut, respectively (reviewed in 50). However, this conclusion is based in part on the prediction that lymphocytes selectively recirculating through an organ will preferentially express adhesion receptors for organ-specific endothelial ligands that allowed their entry into that organ. Thus, when lymphocytes exiting a given tissue expressed high or low levels of a particular adhesion molecule, this has been felt to provide strong support for that molecule being either involved or not involved, respectively, in lymphocyte entry into the tissue. However, the current studies demonstrate a variable and heterogeneous pattern of L-selectin expression on Peyer’s patch lymphocytes in β₇ integrin^−/− mice (Fig. 6) despite the fact that L-selectin is critically involved in lymphocyte migration into this tissue in the absence of β₇ integrin expression (Figs. 3 and 5). Conversely, the majority of PLN lymphocytes express α₄β₇ (27), although the β₇ integrins play no apparent role in lymphocyte migration into PLNs (Figs. 3 and 5). Collectively, these findings force the conclusion that adhesion molecule expression by lymphocytes within or exiting a given tissue is likely to reflect processes or developmental programs ongoing within that tissue rather than emulate the display of adhesion molecules necessary for lymphocyte entry into that tissue.

Although it has been well recognized that L-selectin and the β₇ integrins play major roles in the migration of lymphocytes into secondary lymphoid tissues it was surprising that the loss of these two adhesion molecules eliminated the vast majority of lymphocyte migration into Peyer’s patches. Although concomitant L-selectin/β₇ integrin loss also dramatically inhibited lymphocyte migration into other HEV-bearing lymphoid tissues, PLNs and MLNs retained 7–8% of their wild-type levels of cellularity (Table II), presumably due to lymphocyte entry through their afferent lymphatics. Therefore, whether the deficiency in Peyer’s patches has deleterious or selective effects on mucosal immunity will be an important issue to resolve. Moreover, because lymphocyte entry into PLNs, MLNs, and Peyer’s patches represents only a small portion of lymphocyte migration (51), it will be important to assess whether the loss of L-selectin/β₇ integrins has additional detrimental effects on lymphocyte migration to other extralymphoid sites of lymphocyte accumulation.

We thank Dr. Scott Palmer and Ms. Jennifer Sloane for their help in the early stages of these studies.