The strict regulation of lymphocyte migration to splenic white pulp does not involve common homing receptors

Abstract

Although the spleen is the largest secondary lymphoid organ, little is known about the regulation of lymphocyte migration towards its different compartments of red and white pulp, in contrast to the well-studied mechanisms of lymphocyte homing to lymph nodes. Here we show that short-term trypsin treatment of lymphocytes cleaved off molecules involved in entry into lymph nodes, while homing to the splenic white pulp was unaltered. Prolonged trypsin treatment also abolished the ability of lymphocytes to enter the white pulp. Analysis of affected cell surface molecules and adoptive transfer studies in combination with blocking antibodies revealed that l-selectin, CD44, PSGL-1 and the alpha4 integrins are not required for migration to the white pulp. Although lymphocyte function-associated antigen-1 (LFA-1) is critical for entry into lymph nodes, we show here that in the absence of functional LFA-1 molecules, lymphocytes can still enter the white pulp, in spite of the high expression of intercellular adhesion molecule-1 on sinus lining cells in the marginal zone. The data indicate that adhesion molecules involved in lymphocyte homing to lymph nodes are not essential for migration towards the splenic white pulp, but that additional, trypsin-sensitive, and so far unidentified, molecules are required.

Figure 1

lymphocyte migration to splenic white pulp involves trypsin-sensitive cell surface molecules, that are different from those involved in homing to lymph nodes. (a) Freshly isolated lymphocytes were treated with trypsin for 0, 5, or 45 min, labelled and subsequently injected intravenously into naive recipients (3×10⁷ cells). Transferred cells were visualized 90 min after injection (in red), whereas macrophages in the marginal zone of the spleen and the subcapsular sinus of the lymph node were stained with the anti-sialoadhesin mAb SER-4 (in green), to define the area of the splenic white pulp. Data shown are representative for four independent experiments. (b) Analysis of 45 min trypsinized cells (1×10⁷ cells) 16 hr after injection, indicating that these cells regain the capacity to enter both white pulp and lymph nodes.

Figure 2

Treatment of lymphocytes with trypsin for 5 min does not affect their capacity to enter the white pulp, whereas it completely abrogates lymphocyte migration to lymph nodes. Freshly isolated Ly-5.2⁺ lymphocytes were treated with trypsin for 5 min, mixed in a 1 : 1 ratio with untreated, biotin-labelled Ly-5.2⁺ control cells and this mixture was subsequently injected intravenously into naive Ly-5.1⁺ recipients (6×10⁷ cells per mouse). After 90 min lymphoid organs were collected and the splenic red and white pulp were isolated. Single cell suspensions of the isolated organs were analysed for the ratio of biotinylated versus non-biotinylated cells within the Ly-5.2⁺ population. A ratio of 1 (depicted by horizontal bar) indicates that trypsin-treated cells migrate similarly to control cells. Means and SD are shown for three mice per group.

Figure 3

Chemokine receptor CCR7 is functionally active on 45 min trypsinized lymphocytes: these cells (mainly CD4⁺ T cells) are still able to specifically respond to SLC in a transwell assay. Freshly isolated lymphocytes were treated with trypsin for 0 or 45 min, transferred to a transwell insert (5 µm pore) and subjected to 500 ng/ml SLC in the lower compartment. After 90 min incubation at 37° the amount and phenotype of transmigrated cells was determined by FACS-analysis. (a) The absolute number of transmigrated cells per 10 000 cells, shown with means (horizontal bar) and SD. (b) Percentage of specifically migrated cells, defined as [(no. of cells responding to SLC) − (no. of spontaneously migrating cells)]/(no. of cells applied in the transwell insert) ×100%; data are the mean of six wells and representative for two independent experiments.

Figure 4

Trypsin-sensitivity of various adhesion molecules expressed on naive lymphocytes. Freshly isolated lymphocytes were pretreated with trypsin for 0 min (filled graph), 5 min (solid line) or 45 min (dotted line) and subsequently the expression levels of l-selectin (CD62L), CD44, integrin α₄ (CD49d), integrin α_L (CD11a; LFA-1), integrin β₇ and integrin β₂ (CD18) were determined by FACS-analysis. Expression levels of CD62L, CD44, CD49d and CD11a after 45 min trypsin treatment overlapped with background levels (dotted line; data not shown). Numbers represent the mean fluorescent intensity for each peak. Data are representative for at least four independent experiments.

Figure 5

The effect of blocking antibodies on lymphocyte migration in vivo to the splenic white pulp compared to homing of lymphocytes to peripheral and mesenteric lymph nodes. Freshly isolated Ly-5.2⁺ lymphocytes were pretreated with saturating concentrations of blocking mAb against either integrin α₄, LFA-1, CD44, or PSGL-1 and mixed in a 1 : 1 ratio with biotinylated Ly-5.2⁺ control cells that had been pretreated with non-blocking mAb against the same molecule. This mixture was subsequently injected intravenously into naive Ly-5.1⁺ recipients. After 90 min lymphoid organs were collected and the splenic red and white pulp were isolated. Single-cell suspensions of the isolated organs were analysed for the ratio of biotinylated versus non-biotinylated cells within the Ly-5.2⁺ population; means and SD are shown for three mice per group.

Figure 6

LFA-1^−/− lymphocytes can enter the splenic white pulp, despite the pronounced expression of its ligand, ICAM-1, in the marginal zone. Immunohistological staining of sections from naive spleen show that ICAM-1 is prominently expressed in the marginal zone (a) and colocalizes with MAdCAM-1 on sinus lining cells (data not shown). (b) Splenic sections of wt mice that had been injected with biotinylated LFA-1^−/− lymphocytes show that these cells are able to enter the white pulp within 90 min. The position of the marginal zone is shown here by double staining for MAdCAM-1 with MECA-367 mAb. (c) Isolation of splenic white pulp reveals that LFA-1^−/− and co-injected wt cells can enter this compartment equally well, whereas their entry into lymph nodes is inhibited up to 80%. Means and SD are shown for 3 mice per group.