LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration

Abstract

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1-/-) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-alpha and interleukin-1beta) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1-/- chimeric mice using bone marrow transplantation revealed that in mice with Lsp1-/- endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1-/- neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1-/- animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1-/- mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration.

Figure 1.

The flux of rolling leukocytes (A), rolling cell velocity (B), adherent (C), and emigrated (D) leukocytes in cremasteric venules of TNFα-treated and untreated WT and Lsp1 ^−/− mice. Leukocyte recruitment was induced by intrascrotal injection of TNFα (0.5 μg in 200 μl saline) and the recruitment parameters determined in cremasteric venules from WT (WT control: n = 4; WT + TNFα: n = 3) and Lsp1 ^−/− mice (Lsp1 ^−/− control: n = 6; Lsp1 ^−/− + TNFα: n = 3). *, P < 0.05 and **, P < 0.01, as compared with each untreated control group.

Figure 2.

The flux of rolling leukocytes (A), rolling cell velocity (B), adherent (C), and emigrated (D) leukocytes in cremasteric venules of IL-1β–treated and untreated WT and Lsp1 ^−/− mice. Leukocyte recruitment was induced by intrascrotal injection of IL-1β (12.5 ng in 200 μl saline) and the recruitment parameters determined in cremasteric venules from WT (WT control: n = 4; WT + IL-1β: n = 4) and Lsp1 ^−/− mice (Lsp1 ^−/− control: n = 6; Lsp1 ^−/− + IL-1β: n = 4). *, P < 0.05 and **, P < 0.01, as compared with each untreated control group.

Figure 3.

The flux of rolling leukocytes (A), adherent leukocytes (B), and emigrated leukocytes (C) induced by KC in agarose gel placed 350 μm from the observed cremasteric venule of WT (n = 3) and Lsp1 ^−/− (n = 4) mice. **, P < 0.01 as compared with time 0 (B) or with the WT control (C).

Figure 4.

LSP1 expression in mouse primary lung endothelial cells. (A) RT-PCR analysis of LSP1 mRNA was performed using total RNA extracted from mouse leukocytes and primary lung endothelial cells. (lane M) DNA molecular weight markers (Invitrogen) and (lanes 1–3) RT-PCR products from WT leukocyte RNA extracts (lane 1), Lsp1 ^−/− endothelial RNA extracts (lane 2), and WT endothelial RNA extracts (lane 3). (B and C) Immunoblotting was performed with anti–NH₂-terminal mouse LSP1 (B) and anti–COOH-terminal mouse LSP1 (C) antibodies and with cell extracts as described in Materials and Methods. Cell lysates from 4−8 × 10⁴ leukocytes were loaded in lanes 1 and 2, and 2 × 10⁴ endothelial cells in lanes 3 and 4. Equal protein extracts were loaded in B and C. The protein concentrations in anti–NH₂-terminal LSP1 and anti–COOH-terminal LSP1 solutions used for both blottings were 20 μg/ml. (lane 1) Protein extracts of leukocytes from WT mice; (lane 2) protein extracts of leukocytes from Lsp1 ^−/− mice; (lane 3) protein extracts of lung endothelial cells from WT mice; and (lane 4) protein extracts of lung endothelial cells from Lsp1 ^−/− mice. The numbers to the left are molecular sizes (BenchMark prestained protein ladder; Invitrogen) in kilodaltons. Similar results were observed in three experiments with three batches of endothelial cell isolation.

Figure 5.

Immunofluorescence staining pattern of cultured mouse lung endothelial cells with anti–NH₂-terminal LSP1 and anti–COOH-terminal LSP1. WT (left) and Lsp1 ^−/− (right) mouse lung endothelial cells of passage 1 at subconfluence on glass coverslips were stained with anti–NH₂-terminal LSP1 (top) or anti–COOH-terminal LSP1 (bottom), respectively, as described in Materials and Methods. Magnification, 200.

Figure 6.

Immunofluorescence localization of LSP1 in cultured human endothelial cells. HUVECs cultured on coverslips were double stained for LSP1 (red; A, C, E, and F) and VE-cadherin (green; B and C). (B–D) Cells were also counterstained for DAPI (blue). (C) The overlaid image of A and B. (D) Secondary Ab alone (Texas red) with DAPI staining. (E) The enhanced image (by increasing the contrast) of LSP1 staining pattern. (F) The enhanced image of LSP1 (from E) overlaid with phalloidin (green). Magnification, 400.

Figure 7.

The number of adherent (A and C) and emigrated (B and D) leukocytes in a cremasteric venule of TNFα-treated chimeric mice. WT and Lsp1 ^−/− mice were reconstituted with Lsp1 ^−/− and WT leukocytes, and indicated as Lsp1 ^−/−→WT and WT→Lsp1 ^−/−, respectively (A and B, n = 3). WT and Lsp1 ^−/− mice were also reconstituted with WT and Lsp1 ^−/− leukocytes, and indicated as WT→WT and Lsp1 ^−/−→Lsp1 ^−/−, respectively (C and D, n = 3∼4). Leukocyte recruitment was induced by intrascrotal injection of TNFα (0.5 μg in 200 μl saline) and the recruitment parameters determined in cremasteric venules from these chimeric mice. **, P < 0.01 as compared with the group of Lsp1 ^−/−→WT mice at 4 h. *, P < 0.05 as compared with the group of WT→WT mice at 4 h.

Figure 8.

The number of adherent (A) and emigrated (B) leukocytes induced by KC in an agarose gel placed 350 μm from the observed cremasteric venule of chimeric mice. WT and Lsp1 ^−/− mice were reconstituted with Lsp1 ^−/− leukocytes and WT leukocytes, and indicated as Lsp1 ^−/−→WT (n = 5) and WT→Lsp1 ^−/− (n = 4), respectively. *, P < 0.05 and **, P < 0.01, as compared with time 0 in A, or with the data of the WT mice reconstituted with Lsp1 ^−/− leukocytes in B.

Figure 9.

Microvascular permeability changes in cremasteric venules of WT (n = 4) and Lsp1 ^−/− mice (n = 7) upon histamine superfusion. Measurements were taken before (time 0) and after 0.1 mM histamine superfusion of the cremaster muscle preparation. *, P < 0.05 and **, P < 0.01, as compared with the Lsp1 ^−/− mice at the same time points.