LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration
http://www.100md.com
《实验药学杂志》
1 Immunology Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada
2 Cell and Molecular Biology Division, Toronto Western Research Institute
3 Department of Immunology, University of Toronto, Toronto, Ontario M5T 2S8, Canada
CORRESPONDENCE Paul Kubes: pkubes@ucalgary.ca
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- and interleukin-1?) 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.
Abbreviations used: HUVEC, human umbilical vein endothelial cell; KC, keratinocyte-derived chemokine; LSP1, leukocyte-specific protein 1; Lsp1–/–, LSP1-deficient; MAPK, mitogen-activated protein kinase; MAPKAP, MAPK-activated protein; PKC, protein kinase C.
L. Liu and D.C. Cara contributed equally to this work.
Lymphocyte-specific gene 1, found in both mouse and humans, was initially thought to be restricted to B cells, functional T cells, and thymocytes (1, 2). However, more recently, it has been documented in monocytes, macrophages, and neutrophils and is now referred to as leukocyte-specific protein 1 (LSP1; references 3–5). LSP1 is an intracellular Ca2+ and F-actin binding protein (6–9). In its carboxyl-terminal region, the molecule contains a high affinity F-actin binding site which allows LSP1 to accumulate within the microfilament rich cortical cytoskeleton. LSP1 has been shown to be a major substrate of the mitogen-activated protein kinase (MAPK)-activated protein (MAPKAP) kinase-2 in the p38 MAPK pathway (10). MAPKAP kinase-2 and p38 MAPK were reported to be essential for neutrophil motility and chemotaxis (11–13), suggesting that LSP1 might be important in chemotaxis. However, it should be noted that MAPKAP kinase-2 phosphorylates numerous other molecules, including heat shock protein 25/27 (14), and so the importance of LSP1 after p38 MAPK activation remains unclear. In addition, LSP1 has also been shown to be a substrate for protein kinase C (PKC; 15, 16), which is another molecule implicated in numerous neutrophil functions (including adhesion and chemotaxis; reference 17), which raises the possibility that LSP1 may have multiple roles in neutrophil recruitment.
Although recent in vitro studies using LSP1-deficient (Lsp1–/–) cells suggest that LSP1 does contribute to the process of chemotaxis, as yet unexplained opposing results have been observed. Jongstra-Bilen and colleagues generated Lsp1–/– mice and observed increased chemotactic responses in Lsp1–/– neutrophils in vitro (18, 19). In direct contrast, Hannigan et al. reported, in an in vitro study, reduced chemotactic responses of Lsp1–/– neutrophils, which may be associated with discontinuous primary actin-rich cortexes and large abnormal membrane protrusions (20). Although both groups used keratinocyte-derived chemokine (KC) as a chemoattractant, important differences in experimental conditions between these in vitro experiments included different substrates and different neutrophil populations (peritoneal elicited or bone marrow neutrophils vs. peripheral blood neutrophils). Clearly, a systematic examination of neutrophil function in vivo in the presence and absence of LSP1 is warranted.
In vivo, neutrophil recruitment is a very complex event that requires that neutrophils first tether to the endothelium, and upon activation via chemokines, firmly adhere. This appears to lead to cross-talk between the adherent neutrophil and the endothelium, whereby endothelial cells retract, allowing neutrophils to migrate across the endothelium (21, 22) before chemotaxing toward the source of the injury and/or infection. Recently, we reported that inhibition of p38 MAPK activity dramatically limited neutrophil transmigration across the endothelium and subsequent neutrophil chemotaxis through the interstitium (13). However, whether the p38 MAPK inhibitors were affecting the endothelium and/or the neutrophils was unclear. This is not trivial as both p38 MAPK and MAPKAP kinase-2 have been shown to play a role in endothelial cytoskeletal rearrangements and in increased endothelial permeability associated with hypoxic or oxidative stress (23–25), as well as in TNF or VEGF stimulation (26–28). However, no one to date has assessed the possibility that LSP1 is found in endothelium.
During neutrophil recruitment, the endothelium is thought to actively retract to allow neutrophils to transmigrate (21, 22, 29, 30). Because LSP1 is a major substrate for the p38 MAPK–MAPKAP kinase-2 signaling pathway and MAPK appears to be important in both neutrophils and endothelium, we tested the hypothesis that LSP1 is an important protein in neutrophil extravasation in vivo as a result of endothelial LSP1. Indeed, our data do not support a critical role for neutrophil LSP1 in extravasation in vivo; however, our results reveal that endothelium does have LSP1 and it plays an essential role in transendothelial migration in chimeric mice where LSP1 was selectively expressed in the endothelium.
Results
LSP1 does not affect leukocyte rolling and adhesion, but is important for leukocyte emigration in response to TNF or IL-1?
Table I summarizes the hemodynamics of the microvasculature of Lsp1–/– and WT mice 4 h after intrascrotal injection of TNF. The diameters of the chosen cremasteric venules were similar between WT and Lsp1–/– mice. There was no apparent difference in shear rate, red blood cell velocity (Table I), or calculated blood flow in these postcapillary venules (not depicted). Similarly, during the induction of inflammation, there was a similar decrease in blood flow in postcapillary venules in both WT and Lsp1–/– mice. Therefore, changes in leukocyte behavior described herein cannot be attributed to differences in hemodynamic parameters. There was a small but significant increase in circulating white blood cells in Lsp1–/– mice.
Table I. Hemodynamic parameters in WT and Lsp1–/– mice 4 h after intrascrotal injection of TNF (0.5 μg, n = 3 in each group)
We treated WT and Lsp1–/– mice intrascrotally with 0.5 μg TNF and measured leukocyte rolling flux, rolling velocity, and the number of adherent and emigrated leukocytes in cremasteric venules 3.5, 4, and 4.5 h after cytokine injection. Fig. 1 A demonstrates that 40–60 cells rolled per minute in control preparations of both WT and Lsp1–/– mice. Exposure of the cremaster muscle microcirculation to TNF induced very similar rolling flux in both WT and Lsp1–/– mice. The rolling velocity of leukocytes was 80 μm/s under control conditions in both sets of mice and a very profound 80–90% decrease in rolling velocity was noted in both WT and Lsp1–/– mice after TNF administration (Fig. 1 B). Fig. 1 C demonstrates a large increase in leukocyte adhesion in postcapillary venules after TNF treatment, a response that was again close to identical in WT and Lsp1–/– mice. However, a very significant difference in leukocyte transendothelial migration was noted in WT and Lsp1–/– mice in response to TNF (Fig. 1 D). Although 40 cells emigrated out of vessels per field of view in WT mice, only 15 cells emigrated in Lsp1–/– mice (P < 0.01). In an additional group of WT mice, one fifth the concentration of TNF was used. This caused fewer cells to adhere than in Lsp1–/– mice treated with the higher concentration of TNF, yet the emigration was still higher in WT mice than that in Lsp1–/– mice (unpublished data).
Figure 1. The flux of rolling leukocytes (A), rolling cell velocity (B), adherent (C), and emigrated (D) leukocytes in cremasteric venules of TNF-treated and untreated WT and Lsp1–/– mice. Leukocyte recruitment was induced by intrascrotal injection of TNF (0.5 μg in 200 μl saline) and the recruitment parameters determined in cremasteric venules from WT (WT control: n = 4; WT + TNF: n = 3) and Lsp1–/– mice (Lsp1–/– control: n = 6; Lsp1–/– + TNF: n = 3). *, P < 0.05 and **, P < 0.01, as compared with each untreated control group.
Previous papers have suggested that the mechanisms underlying leukocyte emigration can be quite different for TNF versus, for example, IL-1? (31, 32). To determine whether the impaired emigration was limited to TNF, we injected mice intrascrotally with an optimal dose (12.5 ng) of IL-1? (31), and measured leukocyte rolling flux, rolling velocity, and the number of adherent and emigrated leukocytes in the tissues 3.5, 4, and 4.5 h after injection of cytokine. After IL-1? local administration, leukocyte rolling flux was increased in both WT and Lsp1–/– mice at least twofold greater than untreated control mice (Fig. 2 A). Similar decrease in rolling velocity (Fig. 2 B) and increase in adhesion (Fig. 2 C) to IL-1? was noted in WT and Lsp1–/– mice. Fig. 2 D demonstrates a profound 75% inhibition in leukocyte transendothelial migration in Lsp1–/– mice (P < 0.05). Clearly, LSP1 plays a role in leukocyte emigration in response to proinflammatory cytokines.
Figure 2. The flux of rolling leukocytes (A), rolling cell velocity (B), adherent (C), and emigrated (D) leukocytes in cremasteric venules of IL-1?–treated and untreated WT and Lsp1–/– mice. Leukocyte recruitment was induced by intrascrotal injection of IL-1? (12.5 ng in 200 μl saline) and the recruitment parameters determined in cremasteric venules from WT (WT control: n = 4; WT + IL-1?: n = 4) and Lsp1–/– mice (Lsp1–/– control: n = 6; Lsp1–/– + IL-1?: n = 4). *, P < 0.05 and **, P < 0.01, as compared with each untreated control group.
The impairment in Lsp1–/– mice could be due to an impairment in cytokine signaling and subsequent synthesis of chemokines or it could be an impairment in the emigration process per se. Previous work has demonstrated that essentially all of the emigrated cells at 4-h TNF or IL-1? stimulation are neutrophils (31, 33). Therefore, we examined responses of WT and Lsp1–/– neutrophils after activation of their chemokine receptors to the neutrophil chemoattractant KC in vivo.
LSP1 is essential for neutrophil emigration in response to the chemokine KC
We measured leukocyte rolling, adhesion, and transendothelial migration upon slow release of the chemokine KC from an agarose gel positioned 350 μm from the observed cremasteric postcapillary venule. Rolling was not affected by KC (Fig. 3 A), whereas neutrophils began adhering quite rapidly after the KC-containing gel was placed on the cremaster preparation (Fig. 3 B). However, there was no significant difference in the rolling flux and adhesion response between WT and Lsp1–/– mice. Fig. 3 C summarizes the number of emigrated neutrophils per field of view 60 min after local KC-containing gel addition. In WT animals, 25 neutrophils could be seen outside the venule of study and all of the cells were migrating toward the KC-containing gel (unpublished data). In contrast, in the Lsp1–/– mice, the transendothelial migration was even more impaired in response to the chemokine KC at 60 min than it was in response to cytokines. Less than four cells were seen to migrate across the endothelium.
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).
LSP1 is expressed in mouse and human endothelial cells
Because transendothelial migration is an active process of both leukocytes and endothelium, we isolated leukocytes from the peritoneal cavity in adult mice and primary endothelial cells from the whole lung of 5–7-d-old WT and Lsp1–/– mice. There was insufficient cremaster muscle tissue to harvest sufficient numbers of endothelial cells. The large majority of endothelium isolated from the lung is microvascular in origin. RT-PCR revealed that both WT leukocytes and WT endothelium, but not Lsp1–/– endothelium, had mRNA for LSP1 (Fig. 4 A). By Western blotting and using the original polyclonal anti-LSP1 serum, we observed that WT but not Lsp1–/– mouse primary lung endothelial cells expressed LSP1 protein (52-kD band) and both WT and Lsp1–/– endothelial cells also showed an additional 78 kD band (unpublished data). To obtain more specific antibodies against mouse LSP1, we partially purified the polyclonal anti-LSP1 serum, and by affinity absorption, we made anti-NH2-terminal LSP1 and anti–COOH-terminal LSP1. Fig. 4 shows that both anti–NH2-terminal LSP1 (Fig. 4 B) and anti–COOH-terminal LSP1 (Fig. 4 C) stained the 52-kD LSP1 in WT but not in Lsp1–/– endothelial cells. The molecular mass of the mouse endothelial LSP1 was identical in size to the leukocyte LSP1 (52 kD). This 52-kD band was not observed in endothelial extracts from Lsp1–/– mice (Fig. 4, B and C). Moreover, the band was lost in WT endothelial cells when the LPS1 antibody was preabsorbed against both GST–LSP1 fusion proteins described in Materials and Methods (unpublished data).
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 x 104 leukocytes were loaded in lanes 1 and 2, and 2 x 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.
There was an additional 78-kD band cross-reacting only with anti–NH2-terminal LSP1 antibody expressed in endothelial cells from both WT and Lsp1–/– mice, but not in mouse leukocytes (Fig. 4 B). With two partially purified antibodies against LSP1, we observed fluorescence staining of WT and Lsp1–/– endothelial cells. Anti–NH2-terminal LSP1 antibody stained much brighter on both WT and Lsp1–/– endothelial cells than the anti–COOH-terminal LSP1 staining, and the endothelial cells showed strong cytoskeletal staining with anti–NH2-terminal LSP1 antibody (Fig. 5, top). Because this pattern was also seen in Lsp1–/– mice, this cross-reactivity is likely with another cytoskeletal-associated protein. Anti–COOH-terminal LSP1 antibody specifically stained WT mouse endothelial cells but not Lsp1–/– endothelial cells (Fig. 5, bottom). Interestingly, the distribution of LSP1 in resting WT endothelial cells appeared in the nuclei and very diffusely throughout the cytoplasm. Because LSP1 was stained weakly in the cytoplasm of WT endothelial cells, it was difficult to determine whether LSP1 associated with the endothelial cytoskeleton (Fig. 5, bottom). In Lsp1–/– endothelial cells, the 78-kD protein was still present, but the overall staining was diminished consistent with the lack of LSP1 in Lsp1–/– endothelium.
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.
To determine whether human endothelial cells express LSP1, we double stained human umbilical vein endothelial cells (HUVECs) with anti-LSP1 and anti–VE-cadherin or phalloidin (F-actin). Fig. 6 demonstrates that, similar to mouse endothelial cells, the VE-cadherin–expressing human endothelial cells also express LSP1. Although the majority of LSP1 staining was found in the nucleus, LSP1 staining in the cytoplasm was weak but detectable (Fig. 6 A). Isotype control IgG and secondary Ab alone did not show any significant fluorescence in the nucleus or the cytoplasm (not depicted and Fig. 6 D, respectively). To better detect the LSP1 staining pattern that was relatively weak in the cytoplasm compared with the nuclear staining, we enhanced the fluorescence signals of the dual-labeled LSP1 and phalloidin images (via increasing the contrast; Fig. 6, E and F). The cytoplasmic LSP1 appeared to be distributed throughout the cytoplasm, with some of the cytoplasmic LSP1 overlapping with F-actin (Fig. 6, E and F). Although F-actin formed clear finger-like projections characteristic of cytoskeleton (Fig. 6 F), a significant amount of LSP1 was diffuse and not always associated with these cytoskeletal structures (Fig. 6 E). These results suggest the following: (a) the majority of endothelial LSP1 is nuclear, (b) LSP1 distributes throughout the endothelial cytoplasm, and (c) some endothelial cytoplasmic LSP1 remains associated with F-actin or at least localizes extremely proximate to the endothelial cytoskeleton.
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.
Endothelial LSP1 regulates leukocyte transendothelial migration
The surprising discovery of LSP1 in endothelium led us to ask whether the protein had any function in the dramatic reduction in leukocyte transendothelial migration in Lsp1–/– mice. We made chimeric mice that lacked LSP1 only in the leukocytes. We also made chimeric mice where the Lsp1–/– mice received a BM transplant from WT mice. These mice lack LSP1 in endothelium. Upon TNF local administration, both types of chimeric mice demonstrated similar responsiveness in leukocyte rolling flux (not depicted), rolling velocity (not depicted), and adhesion (Fig. 7 A) in cremasteric venules. Surprisingly, chimeric mice that lacked LSP1 only in their leukocytes emigrated as effectively across the vasculature as WT mice in response to TNF injection, suggesting that the impaired transendothelial emigration was unrelated to leukocyte-derived LSP1 (Fig. 7 B). However, WT leukocytes reconstituted in Lsp1–/– mice (i.e., lacking LSP1 in endothelium) had difficulties in migrating through the Lsp1–/– venules and into the tissue (P < 0.01, as compared with the reversed chimeric mice). Fig. 7 (C and D) demonstrates that WT mice receiving WT BM behaved just like the WT mice in Fig. 1 and Lsp1–/– mice receiving Lsp1–/– BM behaved like Lsp1–/– mice in Fig. 1.
Figure 7. The number of adherent (A and C) and emigrated (B and D) leukocytes in a cremasteric venule of TNF-treated chimeric mice. WT and Lsp1–/– mice were reconstituted with Lsp1–/– and WT leukocytes, and indicated as Lsp1–/–WT and WTLsp1–/–, respectively (A and B, n = 3). WT and Lsp1–/– mice were also reconstituted with WT and Lsp1–/– leukocytes, and indicated as WTWT and Lsp1–/–Lsp1–/–, respectively (C and D, n = 34). Leukocyte recruitment was induced by intrascrotal injection of TNF (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 WTWT mice at 4 h.
In a second series of experiments, we tested responses to KC in the chimeric mice. Both sets of chimeric mice demonstrated similar responsiveness in the leukocyte rolling flux (not depicted), rolling velocity (not depicted), and adhesion (Fig. 8 A) upon placement of the KC-containing gel onto the muscle microvasculature. Again, chimeric mice that lacked LSP1 only in their leukocytes emigrated as effectively across the vasculature as WT mice (Fig. 8 B) in response to KC administration. In contrast, WT leukocytes reconstituted in Lsp1–/– mice (i.e., lacking LSP1 in endothelium) did not display significant transendothelial migration (Fig. 8 B).
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 WTLsp1–/– (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.
LSP1 is important in histamine-stimulated permeability increases in postcapillary venules
This is the first demonstration of LSP1 in endothelium and, more importantly, the first demonstration of a functional role for endothelial LSP1 in regulating leukocyte emigration. Although the mechanism by which Lsp1–/– endothelium restricts leukocyte recruitment is unclear, it is clear that LSP1 is an F-actin binding protein and involved in cytoskeletal changes in leukocytes (7, 8, 19). A very likely possibility is that the Lsp1–/– endothelium did not actively retract to permit leukocyte transendothelial migration. Therefore, we measured permeability responses in the postcapillary venules in the control WT and Lsp1–/– mice. We chose histamine, a stimulus that did not induce neutrophil emigration per se, to avoid complications associated with neutrophils emigrating in WT but not in Lsp1–/– mice. In each case, more FITC-albumin leaked into the interstitium in WT than in Lsp1–/– mice. Fig. 9 shows that at 1, 10, 30, and 60 min after 0.1 mM histamine superfusion, the permeability index in venules of WT mice was significantly higher than in venules of Lsp1–/– mice (P < 0.05). Thus, the results indicated that LSP1 has a functional role in regulating the stimulated permeability changes in postcapillary venules.
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.
LSP1 deficiency does not decrease all forms of leukocyte recruitment
When we administered IL-1? to the peritoneal cavities of WT and Lsp1–/– mice, there was very significant neutrophil recruitment in both strains of mice. In fact, the total leukocytes recovered from the peritoneal lavage were slightly higher in Lsp1–/– mice than in WT mice (at 4 h, 11.1 x 106 ± 1.2 x 106 cells in Lsp1–/– mice vs. 8.0 x 106 ± 0.7 x 106 cells in WT mice; P < 0.05), consistent with previously published results (18).
Discussion
In this paper, we have demonstrated that LSP1 appears to be an essential intracellular molecule involved in the crucial event of transendothelial migration that permits leukocytes to be recruited to sites of inflammation. Neutrophil transendothelial migration across postcapillary venules was clearly impaired in vivo in Lsp1–/– mice when compared with WT littermates. Not all leukocyte functions were inhibited inasmuch as rolling and adhesion within the vasculature (selectin- and integrin-dependent events, respectively) were not altered in Lsp1–/– mice. The impaired recruitment appeared to occur regardless of whether an exogenous chemokine was introduced or whether endogenous chemokines were produced by the local administration of TNF or IL-1?. Until now, LSP1 has been considered to be leukocyte specific; however, we report herein that this F-actin binding protein is also located in microvascular endothelium and functions to permit neutrophil transendothelial migration, suggesting for the first time both a new cellular source of LSP1 and a new critical functional role for LSP1 in endothelium.
Although previous findings of altered neutrophil recruitment in Lsp1–/– mice were considered to exclusively reflect an alteration in neutrophil function per se (18–20), in this paper, we provide evidence that endothelial LSP1 also contributes to the neutrophil recruitment. First, we demonstrated that LSP1 was located in endothelium. The protein was the same size as leukocyte LSP1 and was absent in Lsp1–/– mice. Second, chimeric mice were generated that had WT leukocytes and Lsp1–/– endothelium to delineate the importance of endothelial LSP1. The results clearly demonstrated that WT neutrophils had a profound inability to transmigrate across Lsp1–/– endothelium. In contrast, Lsp1–/– neutrophils migrated across WT endothelium with no impairment. In addition, we report that Lsp1–/– endothelium was less responsive to histamine, a molecule known to activate the endothelial cytoskeleton and induce endothelial retraction.
There is a growing body of evidence that endothelial cells actively contribute to leukocyte transendothelial migration, not only by presenting adhesion molecules including PECAM-1, CD99, and JAM-1, but also by actively retracting and forming gaps upon leukocyte adhesion to the endothelial cells and providing leukocytes a passage of lesser resistance (21, 22, 29, 34, 35). Endothelial cell to cell adherens junctions contain VE-cadherin, which links to different intracellular proteins including ?-catenin, -catenin (plakoglobin), and p120; the former two connect to actin cytoskeleton through the binding to -catenin (36, 37). The retraction process has been shown in vitro and in vivo to involve cytoskeletal rearrangement within the endothelium, leading to the disengagement of catenins and VE-cadherins and to the increase in endothelial permeability (38, 39); this retraction can be triggered by both inflammatory mediators, such as histamine, as well as by the direct process of leukocyte adhesion (21, 22, 29, 34–37, 40). Disruption of endothelial microfilaments significantly reduced leukocyte transmigration (41, 42). Although the signaling events leading to the retraction of the endothelium upstream of the cytoskeleton are not entirely clear, the prevailing view at this stage is that leukocytes engage surface molecules on the endothelium that activate intracellular signaling events, including increased intracellular Ca2+ and activation of PKC and/or myosin light chain kinase, leading to active retraction of endothelium (21, 29, 43, 44). Because LSP1 has actin-binding sites (8, 9), has been shown to play a role in adhesion-dependent polarization (19), and is associated with the cytoplasmic face of the plasma membrane (6), we propose that disruption of LSP1 perturbs the ability of endothelium to retract and, thus, decreases the histamine-induced permeability response of the endothelium and neutrophil extravasation observed in this paper. However, whether LSP1 simply functions upstream of the cytoskeletal changes or physically binds cytoskeleton remains unclear from our immunofluorescence data. It should be noted that overexpression of LSP1 in endothelium (via transfection) revealed significant cytoskeletal association (unpublished data).
It is well appreciated that, in leukocytes, LSP1 is one of several key substrates of MAPKAP kinase-2 and the latter is the direct target of p38 MAPK (10, 45). Several in vitro studies have revealed that both p38 MAPK and MAPKAP kinase-2 are important intracellular signaling molecules in the induction of chemotaxis (11–13). Recently, we have reported that inhibition of p38 MAPK in vivo resulted in both an impairment in transendothelial migration and neutrophil chemotaxis in response to the chemokine KC (13). In contrast, inhibition of p38 MAPK in vitro in human endothelium did not impair recruitment in response to the cytokine TNF (46). In this paper, lack of LSP1 caused impaired emigration in response to both TNF and KC. This clearly suggests that the LSP1 deficiency extends well beyond the role of substrate for p38 MAPK. Indeed, it has been reported that LSP1 is also a major substrate for PKC (15, 16), a molecule known to regulate endothelial retraction. An alternative explanation is that the p38 MAPK inhibitors were less effective than LSP1 deficiency. The p38 MAPK inhibitors used previously in this regard are known to specifically inhibit the p38 and p38? isoform, but not the p38 and p38 isoforms. Because endothelium can express p38, ?, , and (45), if LSP1 is downstream of each of these p38 isoforms, then this may explain the additional inhibition seen in Lsp1–/– mice versus p38 inhibitors.
The nonlethal phenotype of Lsp1–/– mice permitted manipulations that resolved the importance of this molecule in different cell types. Indeed, if the LSP1 pathway is critical within neutrophils for transendothelial migration, then, removal of LSP1 only in leukocytes by generating LSP1 chimeric mice should have still resulted in an inability of Lsp1–/– neutrophils to emigrate out of WT vasculature. Surprisingly, BM transplantation of Lsp1–/– leukocytes into WT mice restored normal transendothelial migration, suggesting that the lack of neutrophil LSP1 was not responsible for the impairment of transendothelial migration in Lsp1–/– mice. Alternative explanations included the possibilities that the irradiation process in some nonspecific manner altered the transendothelial migration phenotype and that the irradiated mice now had an enhanced responsiveness to chemokines. However, our data from the reversed BM chimeras, in which WT neutrophils failed to emigrate out of Lsp1–/– vasculature, refute these alternative explanations and support the view that neutrophil transendothelial migration is dependent on a nonleukocyte source of LSP1.
The impaired neutrophil recruitment due to LSP1 deficiency in our study in muscle appeared to be either stimulus or site specific as the work by Jongstra-Bilen and colleagues clearly revealed the opposite response (i.e., enhanced recruitment of leukocytes into the peritoneal cavity in response to thioglycolate; reference 18). We found that, whereas the recruitment of Lsp1–/– neutrophils was decreased in cremaster muscle upon IL-1? local administration, the recruitment of Lsp1–/– leukocytes into peritoneal cavity was not impaired (but increased) after IL-1? i.p. injection. Although Lsp1–/– neutrophils emigrated less into muscle in response to TNF, we also found that there was no apparent difference in the recruitment of Lsp1–/– and WT leukocytes into peritoneal cavity upon TNF i.p. administration (unpublished data). These results argue against stimulus specificity and support the notion that structural or physiological differences of the organ microvasculatures dictate organ-specific mechanisms of neutrophil recruitment (47).
This is the first documentation of a functional role for LSP1 in endothelium. Some LSP1 could be detected associated with the cytoskeleton; however, the amount was quite low. It may be that the protein only binds the cytoskeleton after leukocyte binding to the endothelium, or perhaps under flow conditions when the endothelium is under constant state of shear force. In addition, under stimulating conditions with a permeabilizing agent, perhaps the movement of LSP1 to cytoskeleton might be observed. Future studies will need to examine under what conditions and how LSP1 interacts with the actin cytoskeleton in endothelium. We will also need to determine whether the recently described function of LSP1 as a cytoskeletal ERK/MAP kinase pathway targeting protein (48) plays a role in its function as an endothelial gatekeeper of leukocyte transendothelial migration.
Materials and Methods
Animals
129/SvJ WT mice were purchased from The Jackson Laboratory. Lsp1–/– mice on the 129/SvJ background were generated by homologous recombination by Jongstra-Bilen and colleagues as described previously (18) and transferred to the University of Calgary Health Sciences Centre. Mice of these two genotypes were bred in the University Animal Centre to obtain age- and sex-matched controls. The mice between 8 and 16 wk of age were used in experiments except for the mice used for isolation of mouse endothelial cells (5–7 d old). TIE2-GFP mice were also purchased from The Jackson Laboratory. All animal protocols were approved by the Animal Care Committee of the University of Calgary and met the standards of the Canadian Association of Animal Care. All animals were kept in specific pathogen-free conditions.
Two types of BM chimeric mice were generated following the standard protocols in our laboratory (33). In brief, BM was isolated from 6–8-wk-old donor mice killed by spinal cord displacement. The BM cell suspensions (8 x 106 cells) from donor Lsp1–/– and 129/SvJ WT mice were injected into the tail vein of 129/SvJ WT and Lsp1–/– mice, respectively. Before BM cell injection, recipients were irradiated with two doses of 5 Gy -ray (Gammacell, 137Cs -irradiation source) with a 3-h interval between the two irradiations. These chimeric mice were housed in specific pathogen-free facilities for 6–8 wk to allow full humoral reconstitution before use in experiments. Initial experiments confirmed that 99% of leukocytes were from donor mice (33). Two additional control groups of mice (WT into WT and Lsp1–/– into Lsp1–/–) went through an identical protocol to ensure that the transplant procedure did not cause any untoward effects.
Intravital microscopy
Male mice were anesthetized with an i.p. injection of a mixture of 10 mg/kg xylazine (Animal Health; Bayer Inc.) and 200 mg/kg ketamine hydrochloride (Rogar/STB Inc.). For all protocols, the left jugular vein was cannulated to administer additional anesthetic or drugs when necessary. The mouse cremaster muscle preparation was used to study the behavior of leukocytes in the microcirculation and adjacent connective tissue as described previously (49). In brief, an incision was made in the scrotal skin to expose the left cremaster muscle, which was then carefully dissected free of the associated fascia. The cremaster muscle was cut longitudinally with a cautery. The testicle and the epididymis were separated from the underlying muscle and were moved into the abdominal cavity. The muscle was held flat on an optically clear viewing pedestal and was secured along the edges with 4–0 suture. The exposed tissue was superfused with 37°C warmed bicarbonate-buffered saline, pH 7.4. An intravital microscope (Axiolskip; Carl Zeiss MicroImaging, Inc.) with a x25 objective lens (Weltzlar L25/0.35; E. Leitz Inc.), and a x10 eyepiece was used to examine the cremasteric microcirculation. A video camera (5100 HS; Panasonic) was used to project the images onto a monitor, and the images were recorded for playback analysis using a videocassette recorder.
Single unbranched cremasteric venules (25–40 μm in diameter) were selected, and to minimize variability, the same section of cremasteric venule was observed throughout the experiment. The number of rolling, adherent, and emigrated leukocytes was determined offline during video playback analysis. Rolling leukocytes were defined as those cells moving at a velocity less than that of erythrocytes within a given vessel. The flux of rolling cells was measured as the number of rolling leukocytes passing by a given point in the venule per minute. Leukocyte rolling velocity was measured for the first 20 leukocytes entering the field of view at the time of recording and calculated from the time required for a leukocyte to roll along a 100-μm length of venule. A leukocyte was considered to be adherent if it remained stationary for at least 30 s, and total leukocyte adhesion was quantified as the number of adherent cells within a 100-μm length of venule in 5 min. Leukocyte emigration was defined as the number of cells in the extravascular space within a 200 x 300-μm area (0.06 mm2), adjacent to the observed venule. More than 90% of these emigrated cells were neutrophils (13, 31). Only cells adjacent to and clearly outside the vessel under study were counted as emigrated.
Induction of leukocyte recruitment in cremaster muscle
To determine whether emigration was impaired in response to cytokines, recombinant mouse TNF (0.5 or 0.1 μg; R&D Systems) or IL-1? (12.5 ng; BD Biosciences) in 200 μl of saline was injected intrascrotally into Lsp1–/–, 129/SvJ WT, or chimeric mice. The leukocyte rolling flux, rolling velocity, adherence, and emigration were measured in the cremasteric venule at 3.5, 4, and 4.5 h after the injection.
To induce neutrophil recruitment independent of cytokines, an agarose gel containing KC (CXCL1; R&D Systems) was used (13, 50). The agarose gel was prepared by adding 10 ml of 2 x HBSS to a boiling concentrated agarose solution (4% in 10 ml of distilled water). A 100-μl aliquot of this solution was removed, and KC was added to this aliquot and mixed to achieve a final concentration of 0.5 μM. To enable visualization of the gel on the cremaster muscle, a small amount of India ink was added to each preparation. A 1-mm3 piece of the mixture (KC-containing gel) was punched out using the tip of a Pasteur pipette. This piece of KC-containing gel was carefully placed on the surface of the cremaster in a preselected area 350 μm (two monitor screens wide) from a postcapillary venule. The gel was held in place using a 22 x 22-mm glass coverslip, and the tissue was superfused beneath the coverslip at a slow rate (0.35 ml/min) to create a chemotactic gradient that permitted emigration. The image was recorded for 90 min: 30 min for control without gel and 60 min with KC-containing gel. In control experiments, only the gel (without KC addition) was placed on the surface of the cremaster muscle.
Microvascular permeability measurement
The degree of vascular albumin leakage from cremasteric venules of Lsp1–/– and control 129/SvJ mice was quantified as described previously (51). In brief, 25 mg/kg FITC-labeled BSA (Sigma-Aldrich) was administered to the mice i.v. at the start of the experiment, and FITC-derived fluorescence (excitation wavelength, 450–490 nm; emission wavelength, 520 nm) was detected using a silicon-intensified charge-coupled device camera (model C-2400-80; Hamamatsu Photonics). Image analysis software (Optimas; Bioscan Inc.) was used to determine the intensity of FITC-albumin–derived fluorescence within the lumen of the venule and in the adjacent perivascular tissue. Background was defined as the fluorescence intensity before FITC-albumin administration. The exposed cremaster muscle was superfused with 0.1 mM histamine dihydrochloride (Sigma-Aldrich) in 37°C warmed bicarbonate-buffered saline. The index of vascular albumin leakage (permeability index) at different time points after histamine superfusion was determined according to the following ratio expressed as a percentage: (mean interstitial intensity – background)/(venular intensity – background) (51).
Harvesting endothelial cells and leukocytes
Acute mouse peritonitis was induced to obtain emigrated leukocytes from Lsp1–/– and control 129/SvJ WT mice. 3 h after an i.p. injection of 1% oyster glycogen (in 1 ml saline; Sigma-Aldrich), leukocytes were lavaged from the peritoneum and prepared for Western blotting (see the next paragraph). Mouse primary lung endothelial cells were isolated from 5–7-d-old Lsp1–/– and control 129/SvJ WT mice, and were cultured according to the protocols described previously (52). Using this protocol with TIE2-GFP mice and flow cytometry, we verified that 93–98% of the isolated cells were GFP positive, confirming that the majority of the purified cells were of endothelial cell origin (53). Freshly isolated mouse endothelial cells were cultured in microvascular endothelial cell medium-2 (Clonetics EGM-2MV BulletKit; Cambrex Bio Science) in 35-mm Petri dishes precoated with 20 μg/ml mouse laminin (Upstate Biotechnology). After reaching confluence in 5–6 d, the cells were either used for Western blotting or trypsinized and subcultured on laminin-coated 22 x 22-mm glass coverslips (at 30,000 cells/coverslip) contained in 35-mm Petri dishes.
Western blot and RT-PCR analysis
The polyclonal anti-LSP1 serum was made in rabbits against mouse recombinant LSP1 protein (6). Although, in leukocytes, the anti-LSP1 serum detected a single band at the appropriate size for LSP1, a second band of 78 kD was detected in endothelium. To remove this reactivity, the GST-LSP1 fusion proteins containing LSP1 residues 1–178 or 179–330 were constructed, subcloned, and expressed in Escherichia coli BL21 (DE3) cells as described previously (54). These two fusion proteins were allowed to conjugate agarose-glutathione beads and were packed into separate glass columns. The polyclonal anti-LSP1 serum was flowed at a rate of <0.15 ml/min (at 4°C) through the different columns containing at least 10-fold molar excess fusion proteins (residues 1–178 or 179–330). The flow-through fractions containing >1.5 mg/ml protein concentrations, which were the anti–COOH-terminal LSP1 and anti–NH2-terminal LSP1, respectively, were collected, pooled separately, and used in Western blotting assays and immunofluorescence microscopy. As a control, the anti-LSP1 serum was also absorbed against both GST-LSP1 fusion proteins and used in Western blotting.
Freshly isolated mouse leukocytes and mouse primary lung endothelial cells were used for Western blot and RT-PCR analysis. Whole cell lysates were prepared from these cells using Laemmli buffer with 10% ?-mercaptoethanol, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. The proteins were separated by electrophoresis in 10% SDS-polyacrylamide gels, transferred to a PVDF Hybond-P transfer membrane (Amersham Biosciences), and blotted using a specific polyclonal rabbit anti–mouse LSP1 serum (at 1:2,000 dilution) as described previously (6) or the anti–COOH-terminal LSP1 and anti–NH2-terminal LSP1 as described before. After washing, the membrane was incubated with a secondary, horseradish peroxidase–conjugated goat anti–rabbit IgG and treated with enhanced chemiluminescence reagents (ECL kit; Amersham Biosciences). The blotted bands were detected with high performance autoradiography films from Amersham Biosciences. RT-PCR was performed using total RNA (100 ng for each cell type) extracted from freshly isolated leukocytes, mouse primary lung endothelial cells, and LSP1 primer pair A1/A4 as described previously (4). The PCR products were electrophoresed by agarose gel, stained with ethidium bromide, and analyzed by high sensitivity Fluor-S Multimager MAX scanner (Bio-Rad Laboratories) upon dark subtraction.
Immunofluorescence microscopy
All rinsing, incubation, and dilution of antibodies was performed in basal buffer that contained 137 NaCl, 5 KCl, 1.1 Na2HPO4, 0.4 KH2PO4, 4 NaHCO3, 5.5 glucose, 4.15 PIPES disodium salt, 2 EGTA, and 4.15 MgCl2 in mM, pH 7.2, at room temperature. Mouse lung primary endothelial cells grown on glass coverslips for 24 h were fixed with 4% formalin, permeabilized with 0.1% Triton X-100, and incubated with 10 μg/ml glycine. Primary antibodies used were the anti–COOH-terminal LSP1 and anti–NH2-terminal LSP1 as described before for 30 min where the protein concentrations were both 0.4 mg/ml in the two blotting solutions, or the original rabbit polyclonal anti-LSP1 serum (at 1:100 dilution). After rinsing three times with 0.1% Tween-20, the coverslips were incubated with Cy3-conjugated goat anti–rabbit IgG for 30 min. After rinsing with basal buffer, the coverslips were mounted in 90% glycerol. Observations were performed on an Olympus IX-70 fluorescence microscope (Olympus). Fluorescence images were captured using OpenLab software (version 3.1.5; Improvision Inc.).
To determine whether human endothelial cells express LSP1, we isolated and cultured HUVECs as described previously (46). Once the HUVECs were confluent, they were passaged onto fibronectin-coated glass coverslips. After culture for 24–48 h, these HUVECs were stained as outlined before with 2.5 μg/ml mouse anti–human LSP1 mAb (clone 16; BD Biosciences) and goat anti–mouse IgG conjugated with Texas Red (Molecular Probes). DAPI (4',6-diamidino-2-phenylindole, dihydrochloride; Molecular Probes) was used for nuclear staining. To confirm that endothelial cells and not a contaminating cell type expressed LSP1, we dual labeled the HUVECs with FITC-conjugated anti–human VE-cadherin (Bender Medsystems) in combination with LSP1 staining. To assess the association of LSP1 with the cytoskeleton, we labeled HUVECs with anti-LSP1 and phalloidin (Alexa Fluor conjugated; Molecular Probes).
Statistical analysis
The data are expressed as means ± SEM. A Student's t test was applied to compare the statistical difference within two groups, and analysis of variance was used for the comparison of the differences in more than two groups. A p-value of <0.05 was considered statistically significant.
Acknowledgments
We thank L. Zbytnuik and K. Jorgensen for their expert assistance in animal care.
This work was supported in part by a Canadian Institutes of Health Research group grant and by the Heart and Stroke Foundation of Canada. L. Liu is supported by a fellowship from Heart and Stroke Foundation of Canada and Alberta Heritage Foundation for Medical Research. D.C. Cara has a postdoctoral fellowship from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq, Brasilia, Brazil. P. Kubes is an Alberta Heritage Foundation for Medical Research Scientist and a Canada Research Chair.
References
Jongstra, J., G.F. Tidmarsh, J. Jongstra-Bilen, and M.M. Davis. 1988. A new lymphocyte-specific gene which encodes a putative Ca2+-binding protein is not expressed in transformed T lymphocyte lines. J. Immunol. 141:3999–4004.
Jongstra-Bilen, J., A.J. Young, R. Chong, and J. Jongstra. 1990. Human and mouse LSP1 genes code for highly conserved phosphoproteins. J. Immunol. 144:1104–1110.
Pulford, K., M. Jones, A.H. Banham, E. Haralambieva, and D.Y. Mason. 1999. Lymphocyte-specific protein 1: a specific marker of human leucocytes. Immunology. 96:262–271.
Jongstra, J., M.E. Ittel, N.N. Iscove, and G. Brady. 1994. The LSP1 gene is expressed in cultured normal and transformed mouse macrophages. Mol. Immunol. 31:1125–1131.
Li, Y., A. Guerrero, and T.H. Howard. 1995. The actin-binding protein, lymphocyte-specific protein 1, is expressed in human leukocytes and human myeloid and lymphoid cell lines. J. Immunol. 155:3563–3569.
Klein, D.P., J. Jongstra-Bilen, K. Ogryzlo, R. Chong, and J. Jongstra. 1989. Lymphocyte-specific Ca2+-binding protein LSP1 is associated with the cytoplasmic face of the plasma membrane. Mol. Cell. Biol. 9:3043–3048.
Klein, D.P., S. Galea, and J. Jongstra. 1990. The lymphocyte-specific protein LSP1 is associated with the cytoskeleton and co-caps with membrane IgM. J. Immunol. 145:2967–2973.
Jongstra-Bilen, J., P.A. Janmey, J.H. Hartwig, S. Galea, and J. Jongstra. 1992. The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain. J. Cell Biol. 118:1443–1453.
Zhang, Q., Y. Li, and T.H. Howard. 2000. Human lymphocyte-specific protein 1, the protein overexpressed in neutrophil actin dysfunction with 47-kDa and 89-kDa protein abnormalities (NAD 47/89), has multiple F-actin binding domains. J. Immunol. 165:2052–2058.
Huang, C.K., L. Zhan, Y. Ai, and J. Jongstra. 1997. LSP1 is the major substrate for mitogen-activated protein kinase-activated protein kinase 2 in human neutrophils. J. Biol. Chem. 272:17–19.
Hannigan, M.O., L. Zhan, Y. Ai, A. Kotlyarov, M. Gaestel, and C.K. Huang. 2001. Abnormal migration phenotype of mitogen-activated protein kinase-activated protein kinase 2–/– neutrophils in Zigmond chambers containing formyl-methionyl-leucyl-phenylalanine gradients. J. Immunol. 167:3953–3961.
Zu, Y.L., J. Qi, A. Gilchrist, G.A. Fernandez, D. Vazquez-Abad, D.L. Kreutzer, C.K. Huang, and R.I. Sha'afi. 1998. p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF- or FMLP stimulation. J. Immunol. 160:1982–1989.
Cara, D.C., J. Kaur, M. Forster, D.M. McCafferty, and P. Kubes. 2001. Role of p38 mitogen-activated protein kinase in chemokine-induced emigration and chemotaxis in vivo. J. Immunol. 167:6552–6558.
Stokoe, D., K. Engel, D.G. Campbell, P. Cohen, and M. Gaestel. 1992. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett. 313:307–313.
Carballo, E., D. Colomer, J.L. Vives-Corrons, P.J. Blackshear, and J. Gil. 1996. Characterization and purification of a protein kinase C substrate in human B cells. Identification as lymphocyte-specific protein 1 (LSP1). J. Immunol. 156:1709–1713.
Matsumoto, N., S. Kojima, T. Osawa, and S. Toyoshima. 1995. Protein kinase C phosphorylates p50 LSP1 and induces translocation of p50 LSP1 in T lymphocytes. J. Biochem. (Tokyo). 117:222–229.
Laudanna, C., D. Mochly-Rosen, T. Liron, G. Constantin, and E.C. Butcher. 1998. Evidence of protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis. J. Biol. Chem. 273:30306–30315.
Jongstra-Bilen, J., V.L. Misener, C. Wang, H. Ginzberg, A. Auerbach, A.L. Joyner, G.P. Downey, and J. Jongstra. 2000. LSP1 modulates leukocyte populations in resting and inflamed peritoneum. Blood. 96:1827–1835.
Wang, C., H. Hayashi, R. Harrison, B. Chiu, J.R. Chan, H.L. Ostergaard, R.D. Inman, J. Jongstra, M.I. Cybulsky, and J. Jongstra-Bilen. 2002. Modulation of Mac-1 (CD11b/CD18)-mediated adhesion by the leukocyte-specific protein 1 is key to its role in neutrophil polarization and chemotaxis. J. Immunol. 169:415–423.
Hannigan, M., L. Zhan, Y. Ai, and C.K. Huang. 2001. Leukocyte-specific gene 1 protein (LSP1) is involved in chemokine KC-activated cytoskeletal reorganization in murine neutrophils in vitro. J. Leukoc. Biol. 69:497–504.
Luscinskas, F.W., S. Ma, A. Nusrat, C.A. Parkos, and S.K. Shaw. 2002. Leukocyte transendothelial migration: a junctional affair. Semin. Immunol. 14:105–113.
Vestweber, D. 2002. Regulation of endothelial cell contacts during leukocyte extravasation. Curr. Opin. Cell Biol. 14:587–593.
Kayyali, U.S., C.M. Pennella, C. Trujillo, O. Villa, M. Gaestel, and P.M. Hassoun. 2002. Cytoskeletal changes in hypoxic pulmonary endothelial cells are dependent on MAPK-activated protein kinase MK2. J. Biol. Chem. 277:42596–42602.
Park, J.H., N. Okayama, D. Gute, A. Krsmanovic, H. Battarbee, and J.S. Alexander. 1999. Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton. Am. J. Physiol. 277:C1066–C1074.
Huot, J., F. Houle, F. Marceau, and J. Landry. 1997. Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ. Res. 80:383–392.
Nwariaku, F.E., J. Chang, X. Zhu, Z. Liu, S.L. Duffy, N.H. Halaihel, L. Terada, and R.H. Turnage. 2002. The role of p38 MAP kinase in tumor necrosis factor-induced redistribution of vascular endothelial cadherin and increased endothelial permeability. Shock. 18:82–85.
Laird, S.M., A. Graham, A. Paul, G.W. Gould, C. Kennedy, and R. Plevin. 1998. Tumour necrosis factor stimulates stress-activated protein kinases and the inhibition of DNA synthesis in cultures of bovine aortic endothelial cells. Cell. Signal. 10:473–480.
Rousseau, S., F. Houle, J. Landry, and J. Huot. 1997. p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene. 15:2169–2177.
Huang, A.J., J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells. J. Cell Biol. 120:1371–1380.
Garcia, J.G., A.D. Verin, M. Herenyiova, and D. English. 1998. Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration. J. Appl. Physiol. 84:1817–1821.
Thompson, R.D., K.E. Noble, K.Y. Larbi, A. Dewar, G.S. Duncan, T.W. Mak, and S. Nourshargh. 2001. Platelet-endothelial cell adhesion molecule-1 (PECAM-1)–deficient mice demonstrate a transient and cytokine-specific role for PECAM-1 in leukocyte migration through the perivascular basement membrane. Blood. 97:1854–1860.
Young, R.E., R.D. Thompson, and S. Nourshargh. 2002. Divergent mechanisms of action of the inflammatory cytokines interleukin 1-? and tumour necrosis factor- in mouse cremasteric venules. Br. J. Pharmacol. 137:1237–1246.
Carvalho-Tavares, J., M.J. Hickey, J. Hutchison, J. Michaud, I.T. Sutcliffe, and P. Kubes. 2000. A role for platelets and endothelial selectins in tumor necrosis factor--induced leukocyte recruitment in the brain microvasculature. Circ. Res. 87:1141–1148.
Del Maschio, A., A. Zanetti, M. Corada, Y. Rival, L. Ruco, M.G. Lampugnani, and E. Dejana. 1996. Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions. J. Cell Biol. 135:497–510.
Johnson-Leger, C., M. Aurrand-Lions, and B.A. Imhof. 2000. The parting of the endothelium: miracle, or simply a junctional affair? J. Cell Sci. 113:921–933.
Dejana, E. 1996. Endothelial adherens junctions: implications in the control of vascular permeability and angiogenesis. J. Clin. Invest. 98:1949–1953.
Dejana, E., R. Spagnuolo, and G. Bazzoni. 2001. Interendothelial junctions and their role in the control of angiogenesis, vascular permeability and leukocyte transmigration. Thromb. Haemost. 86:308–315.
Waschke, J., W. Baumgartner, R.H. Adamson, M. Zeng, K. Aktories, H. Barth, C. Wilde, F.E. Curry, and D. Drenckhahn. 2004. Requirement of Rac activity for maintenance of capillary endothelial barrier properties. Am. J. Physiol. Heart Circ. Physiol. 286:H394–H401.
Adamson, R.H., F.E. Curry, G. Adamson, B. Liu, Y. Jiang, K. Aktories, H. Barth, A. Daigeler, N. Golenhofen, W. Ness, and D. Drenckhahn. 2002. Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice. J. Physiol. 539:295–308.
Shaw, S.K., P.S. Bamba, B.N. Perkins, and F.W. Luscinskas. 2001. Real-time imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium. J. Immunol. 167:2323–2330.
Kielbassa, K., C. Schmitz, and V. Gerke. 1998. Disruption of endothelial microfilaments selectively reduces the transendothelial migration of monocytes. Exp. Cell Res. 243:129–141.
Hordijk, P.L., E. Anthony, F.P. Mul, R. Rientsma, L.C. Oomen, and D. Roos. 1999. Vascular-endothelial-cadherin modulates endothelial monolayer permeability. J. Cell Sci. 112:1915–1923.
Su, W.H., H.I. Chen, J.P. Huang, and C.J. Jen. 2000. Endothelial i signaling during transmigration of polymorphonuclear leukocytes. Blood. 96:3816–3822.
Lum, H., and A.B. Malik. 1994. Regulation of vascular endothelial barrier function. Am. J. Physiol. 267:L223–L241.
Herlaar, E., and Z. Brown. 1999. p38 MAPK signalling cascades in inflammatory disease. Mol. Med. Today. 5:439–447.
Kaur, J., R.C. Woodman, and P. Kubes. 2003. P38 MAPK: critical molecule in thrombin-induced NF-B-dependent leukocyte recruitment. Am. J. Physiol. Heart Circ. Physiol. 284:H1095–H1103.
Liu, L., and P. Kubes. 2003. Molecular mechanisms of leukocyte recruitment: organ-specific mechanisms of action. Thromb. Haemost. 89:213–220.
Harrison, R.E., B.A. Sikorski, and J. Jongstra. 2004. Leukocyte-specific protein 1 targets the ERK/MAP kinase scaffold protein KSR and MEK1 and ERK2 to the actin cytoskeleton. J. Cell Sci. 117:2151–2157.
Kanwar, S., D.C. Bullard, M.J. Hickey, C.W. Smith, A.L. Beaudet, B.A. Wolitzky, and P. Kubes. 1997. The association between 4-integrin, P-selectin, and E-selectin in an allergic model of inflammation. J. Exp. Med. 185:1077–1087.
Hickey, M.J., M. Forster, D. Mitchell, J. Kaur, C. De Caigny, and P. Kubes. 2000. L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo. J. Immunol. 165:7164–7170.
Kurose, I., P. Kubes, R. Wolf, D.C. Anderson, J. Paulson, M. Miyasaka, and D.N. Granger. 1993. Inhibition of nitric oxide production. Mechanisms of vascular albumin leakage. Circ. Res. 73:164–171.
Bowden, R.A., Z.M. Ding, E.M. Donnachie, T.K. Petersen, L.H. Michael, C.M. Ballantyne, and A.R. Burns. 2002. Role of 4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium. Circ. Res. 90:562–569.
Motoike, T., S. Loughna, E. Perens, B.L. Roman, W. Liao, T.C. Chau, C.D. Richardson, T. Kawate, J. Kuno, B.M. Weinstein, et al. 2000. Universal GFP reporter for the study of vascular development. Genesis. 28:75–81.
Wong, M.J., I.A. Malapitan, B.A. Sikorski, and J. Jongstra. 2003. A cell-free binding assay maps the LSP1 cytoskeletal binding site to the COOH-terminal 30 amino acids. Biochim. Biophys. Acta. 1642:17–24.(Lixin Liu1, Denise C. Car)
2 Cell and Molecular Biology Division, Toronto Western Research Institute
3 Department of Immunology, University of Toronto, Toronto, Ontario M5T 2S8, Canada
CORRESPONDENCE Paul Kubes: pkubes@ucalgary.ca
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- and interleukin-1?) 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.
Abbreviations used: HUVEC, human umbilical vein endothelial cell; KC, keratinocyte-derived chemokine; LSP1, leukocyte-specific protein 1; Lsp1–/–, LSP1-deficient; MAPK, mitogen-activated protein kinase; MAPKAP, MAPK-activated protein; PKC, protein kinase C.
L. Liu and D.C. Cara contributed equally to this work.
Lymphocyte-specific gene 1, found in both mouse and humans, was initially thought to be restricted to B cells, functional T cells, and thymocytes (1, 2). However, more recently, it has been documented in monocytes, macrophages, and neutrophils and is now referred to as leukocyte-specific protein 1 (LSP1; references 3–5). LSP1 is an intracellular Ca2+ and F-actin binding protein (6–9). In its carboxyl-terminal region, the molecule contains a high affinity F-actin binding site which allows LSP1 to accumulate within the microfilament rich cortical cytoskeleton. LSP1 has been shown to be a major substrate of the mitogen-activated protein kinase (MAPK)-activated protein (MAPKAP) kinase-2 in the p38 MAPK pathway (10). MAPKAP kinase-2 and p38 MAPK were reported to be essential for neutrophil motility and chemotaxis (11–13), suggesting that LSP1 might be important in chemotaxis. However, it should be noted that MAPKAP kinase-2 phosphorylates numerous other molecules, including heat shock protein 25/27 (14), and so the importance of LSP1 after p38 MAPK activation remains unclear. In addition, LSP1 has also been shown to be a substrate for protein kinase C (PKC; 15, 16), which is another molecule implicated in numerous neutrophil functions (including adhesion and chemotaxis; reference 17), which raises the possibility that LSP1 may have multiple roles in neutrophil recruitment.
Although recent in vitro studies using LSP1-deficient (Lsp1–/–) cells suggest that LSP1 does contribute to the process of chemotaxis, as yet unexplained opposing results have been observed. Jongstra-Bilen and colleagues generated Lsp1–/– mice and observed increased chemotactic responses in Lsp1–/– neutrophils in vitro (18, 19). In direct contrast, Hannigan et al. reported, in an in vitro study, reduced chemotactic responses of Lsp1–/– neutrophils, which may be associated with discontinuous primary actin-rich cortexes and large abnormal membrane protrusions (20). Although both groups used keratinocyte-derived chemokine (KC) as a chemoattractant, important differences in experimental conditions between these in vitro experiments included different substrates and different neutrophil populations (peritoneal elicited or bone marrow neutrophils vs. peripheral blood neutrophils). Clearly, a systematic examination of neutrophil function in vivo in the presence and absence of LSP1 is warranted.
In vivo, neutrophil recruitment is a very complex event that requires that neutrophils first tether to the endothelium, and upon activation via chemokines, firmly adhere. This appears to lead to cross-talk between the adherent neutrophil and the endothelium, whereby endothelial cells retract, allowing neutrophils to migrate across the endothelium (21, 22) before chemotaxing toward the source of the injury and/or infection. Recently, we reported that inhibition of p38 MAPK activity dramatically limited neutrophil transmigration across the endothelium and subsequent neutrophil chemotaxis through the interstitium (13). However, whether the p38 MAPK inhibitors were affecting the endothelium and/or the neutrophils was unclear. This is not trivial as both p38 MAPK and MAPKAP kinase-2 have been shown to play a role in endothelial cytoskeletal rearrangements and in increased endothelial permeability associated with hypoxic or oxidative stress (23–25), as well as in TNF or VEGF stimulation (26–28). However, no one to date has assessed the possibility that LSP1 is found in endothelium.
During neutrophil recruitment, the endothelium is thought to actively retract to allow neutrophils to transmigrate (21, 22, 29, 30). Because LSP1 is a major substrate for the p38 MAPK–MAPKAP kinase-2 signaling pathway and MAPK appears to be important in both neutrophils and endothelium, we tested the hypothesis that LSP1 is an important protein in neutrophil extravasation in vivo as a result of endothelial LSP1. Indeed, our data do not support a critical role for neutrophil LSP1 in extravasation in vivo; however, our results reveal that endothelium does have LSP1 and it plays an essential role in transendothelial migration in chimeric mice where LSP1 was selectively expressed in the endothelium.
Results
LSP1 does not affect leukocyte rolling and adhesion, but is important for leukocyte emigration in response to TNF or IL-1?
Table I summarizes the hemodynamics of the microvasculature of Lsp1–/– and WT mice 4 h after intrascrotal injection of TNF. The diameters of the chosen cremasteric venules were similar between WT and Lsp1–/– mice. There was no apparent difference in shear rate, red blood cell velocity (Table I), or calculated blood flow in these postcapillary venules (not depicted). Similarly, during the induction of inflammation, there was a similar decrease in blood flow in postcapillary venules in both WT and Lsp1–/– mice. Therefore, changes in leukocyte behavior described herein cannot be attributed to differences in hemodynamic parameters. There was a small but significant increase in circulating white blood cells in Lsp1–/– mice.
Table I. Hemodynamic parameters in WT and Lsp1–/– mice 4 h after intrascrotal injection of TNF (0.5 μg, n = 3 in each group)
We treated WT and Lsp1–/– mice intrascrotally with 0.5 μg TNF and measured leukocyte rolling flux, rolling velocity, and the number of adherent and emigrated leukocytes in cremasteric venules 3.5, 4, and 4.5 h after cytokine injection. Fig. 1 A demonstrates that 40–60 cells rolled per minute in control preparations of both WT and Lsp1–/– mice. Exposure of the cremaster muscle microcirculation to TNF induced very similar rolling flux in both WT and Lsp1–/– mice. The rolling velocity of leukocytes was 80 μm/s under control conditions in both sets of mice and a very profound 80–90% decrease in rolling velocity was noted in both WT and Lsp1–/– mice after TNF administration (Fig. 1 B). Fig. 1 C demonstrates a large increase in leukocyte adhesion in postcapillary venules after TNF treatment, a response that was again close to identical in WT and Lsp1–/– mice. However, a very significant difference in leukocyte transendothelial migration was noted in WT and Lsp1–/– mice in response to TNF (Fig. 1 D). Although 40 cells emigrated out of vessels per field of view in WT mice, only 15 cells emigrated in Lsp1–/– mice (P < 0.01). In an additional group of WT mice, one fifth the concentration of TNF was used. This caused fewer cells to adhere than in Lsp1–/– mice treated with the higher concentration of TNF, yet the emigration was still higher in WT mice than that in Lsp1–/– mice (unpublished data).
Figure 1. The flux of rolling leukocytes (A), rolling cell velocity (B), adherent (C), and emigrated (D) leukocytes in cremasteric venules of TNF-treated and untreated WT and Lsp1–/– mice. Leukocyte recruitment was induced by intrascrotal injection of TNF (0.5 μg in 200 μl saline) and the recruitment parameters determined in cremasteric venules from WT (WT control: n = 4; WT + TNF: n = 3) and Lsp1–/– mice (Lsp1–/– control: n = 6; Lsp1–/– + TNF: n = 3). *, P < 0.05 and **, P < 0.01, as compared with each untreated control group.
Previous papers have suggested that the mechanisms underlying leukocyte emigration can be quite different for TNF versus, for example, IL-1? (31, 32). To determine whether the impaired emigration was limited to TNF, we injected mice intrascrotally with an optimal dose (12.5 ng) of IL-1? (31), and measured leukocyte rolling flux, rolling velocity, and the number of adherent and emigrated leukocytes in the tissues 3.5, 4, and 4.5 h after injection of cytokine. After IL-1? local administration, leukocyte rolling flux was increased in both WT and Lsp1–/– mice at least twofold greater than untreated control mice (Fig. 2 A). Similar decrease in rolling velocity (Fig. 2 B) and increase in adhesion (Fig. 2 C) to IL-1? was noted in WT and Lsp1–/– mice. Fig. 2 D demonstrates a profound 75% inhibition in leukocyte transendothelial migration in Lsp1–/– mice (P < 0.05). Clearly, LSP1 plays a role in leukocyte emigration in response to proinflammatory cytokines.
Figure 2. The flux of rolling leukocytes (A), rolling cell velocity (B), adherent (C), and emigrated (D) leukocytes in cremasteric venules of IL-1?–treated and untreated WT and Lsp1–/– mice. Leukocyte recruitment was induced by intrascrotal injection of IL-1? (12.5 ng in 200 μl saline) and the recruitment parameters determined in cremasteric venules from WT (WT control: n = 4; WT + IL-1?: n = 4) and Lsp1–/– mice (Lsp1–/– control: n = 6; Lsp1–/– + IL-1?: n = 4). *, P < 0.05 and **, P < 0.01, as compared with each untreated control group.
The impairment in Lsp1–/– mice could be due to an impairment in cytokine signaling and subsequent synthesis of chemokines or it could be an impairment in the emigration process per se. Previous work has demonstrated that essentially all of the emigrated cells at 4-h TNF or IL-1? stimulation are neutrophils (31, 33). Therefore, we examined responses of WT and Lsp1–/– neutrophils after activation of their chemokine receptors to the neutrophil chemoattractant KC in vivo.
LSP1 is essential for neutrophil emigration in response to the chemokine KC
We measured leukocyte rolling, adhesion, and transendothelial migration upon slow release of the chemokine KC from an agarose gel positioned 350 μm from the observed cremasteric postcapillary venule. Rolling was not affected by KC (Fig. 3 A), whereas neutrophils began adhering quite rapidly after the KC-containing gel was placed on the cremaster preparation (Fig. 3 B). However, there was no significant difference in the rolling flux and adhesion response between WT and Lsp1–/– mice. Fig. 3 C summarizes the number of emigrated neutrophils per field of view 60 min after local KC-containing gel addition. In WT animals, 25 neutrophils could be seen outside the venule of study and all of the cells were migrating toward the KC-containing gel (unpublished data). In contrast, in the Lsp1–/– mice, the transendothelial migration was even more impaired in response to the chemokine KC at 60 min than it was in response to cytokines. Less than four cells were seen to migrate across the endothelium.
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).
LSP1 is expressed in mouse and human endothelial cells
Because transendothelial migration is an active process of both leukocytes and endothelium, we isolated leukocytes from the peritoneal cavity in adult mice and primary endothelial cells from the whole lung of 5–7-d-old WT and Lsp1–/– mice. There was insufficient cremaster muscle tissue to harvest sufficient numbers of endothelial cells. The large majority of endothelium isolated from the lung is microvascular in origin. RT-PCR revealed that both WT leukocytes and WT endothelium, but not Lsp1–/– endothelium, had mRNA for LSP1 (Fig. 4 A). By Western blotting and using the original polyclonal anti-LSP1 serum, we observed that WT but not Lsp1–/– mouse primary lung endothelial cells expressed LSP1 protein (52-kD band) and both WT and Lsp1–/– endothelial cells also showed an additional 78 kD band (unpublished data). To obtain more specific antibodies against mouse LSP1, we partially purified the polyclonal anti-LSP1 serum, and by affinity absorption, we made anti-NH2-terminal LSP1 and anti–COOH-terminal LSP1. Fig. 4 shows that both anti–NH2-terminal LSP1 (Fig. 4 B) and anti–COOH-terminal LSP1 (Fig. 4 C) stained the 52-kD LSP1 in WT but not in Lsp1–/– endothelial cells. The molecular mass of the mouse endothelial LSP1 was identical in size to the leukocyte LSP1 (52 kD). This 52-kD band was not observed in endothelial extracts from Lsp1–/– mice (Fig. 4, B and C). Moreover, the band was lost in WT endothelial cells when the LPS1 antibody was preabsorbed against both GST–LSP1 fusion proteins described in Materials and Methods (unpublished data).
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 x 104 leukocytes were loaded in lanes 1 and 2, and 2 x 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.
There was an additional 78-kD band cross-reacting only with anti–NH2-terminal LSP1 antibody expressed in endothelial cells from both WT and Lsp1–/– mice, but not in mouse leukocytes (Fig. 4 B). With two partially purified antibodies against LSP1, we observed fluorescence staining of WT and Lsp1–/– endothelial cells. Anti–NH2-terminal LSP1 antibody stained much brighter on both WT and Lsp1–/– endothelial cells than the anti–COOH-terminal LSP1 staining, and the endothelial cells showed strong cytoskeletal staining with anti–NH2-terminal LSP1 antibody (Fig. 5, top). Because this pattern was also seen in Lsp1–/– mice, this cross-reactivity is likely with another cytoskeletal-associated protein. Anti–COOH-terminal LSP1 antibody specifically stained WT mouse endothelial cells but not Lsp1–/– endothelial cells (Fig. 5, bottom). Interestingly, the distribution of LSP1 in resting WT endothelial cells appeared in the nuclei and very diffusely throughout the cytoplasm. Because LSP1 was stained weakly in the cytoplasm of WT endothelial cells, it was difficult to determine whether LSP1 associated with the endothelial cytoskeleton (Fig. 5, bottom). In Lsp1–/– endothelial cells, the 78-kD protein was still present, but the overall staining was diminished consistent with the lack of LSP1 in Lsp1–/– endothelium.
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.
To determine whether human endothelial cells express LSP1, we double stained human umbilical vein endothelial cells (HUVECs) with anti-LSP1 and anti–VE-cadherin or phalloidin (F-actin). Fig. 6 demonstrates that, similar to mouse endothelial cells, the VE-cadherin–expressing human endothelial cells also express LSP1. Although the majority of LSP1 staining was found in the nucleus, LSP1 staining in the cytoplasm was weak but detectable (Fig. 6 A). Isotype control IgG and secondary Ab alone did not show any significant fluorescence in the nucleus or the cytoplasm (not depicted and Fig. 6 D, respectively). To better detect the LSP1 staining pattern that was relatively weak in the cytoplasm compared with the nuclear staining, we enhanced the fluorescence signals of the dual-labeled LSP1 and phalloidin images (via increasing the contrast; Fig. 6, E and F). The cytoplasmic LSP1 appeared to be distributed throughout the cytoplasm, with some of the cytoplasmic LSP1 overlapping with F-actin (Fig. 6, E and F). Although F-actin formed clear finger-like projections characteristic of cytoskeleton (Fig. 6 F), a significant amount of LSP1 was diffuse and not always associated with these cytoskeletal structures (Fig. 6 E). These results suggest the following: (a) the majority of endothelial LSP1 is nuclear, (b) LSP1 distributes throughout the endothelial cytoplasm, and (c) some endothelial cytoplasmic LSP1 remains associated with F-actin or at least localizes extremely proximate to the endothelial cytoskeleton.
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.
Endothelial LSP1 regulates leukocyte transendothelial migration
The surprising discovery of LSP1 in endothelium led us to ask whether the protein had any function in the dramatic reduction in leukocyte transendothelial migration in Lsp1–/– mice. We made chimeric mice that lacked LSP1 only in the leukocytes. We also made chimeric mice where the Lsp1–/– mice received a BM transplant from WT mice. These mice lack LSP1 in endothelium. Upon TNF local administration, both types of chimeric mice demonstrated similar responsiveness in leukocyte rolling flux (not depicted), rolling velocity (not depicted), and adhesion (Fig. 7 A) in cremasteric venules. Surprisingly, chimeric mice that lacked LSP1 only in their leukocytes emigrated as effectively across the vasculature as WT mice in response to TNF injection, suggesting that the impaired transendothelial emigration was unrelated to leukocyte-derived LSP1 (Fig. 7 B). However, WT leukocytes reconstituted in Lsp1–/– mice (i.e., lacking LSP1 in endothelium) had difficulties in migrating through the Lsp1–/– venules and into the tissue (P < 0.01, as compared with the reversed chimeric mice). Fig. 7 (C and D) demonstrates that WT mice receiving WT BM behaved just like the WT mice in Fig. 1 and Lsp1–/– mice receiving Lsp1–/– BM behaved like Lsp1–/– mice in Fig. 1.
Figure 7. The number of adherent (A and C) and emigrated (B and D) leukocytes in a cremasteric venule of TNF-treated chimeric mice. WT and Lsp1–/– mice were reconstituted with Lsp1–/– and WT leukocytes, and indicated as Lsp1–/–WT and WTLsp1–/–, respectively (A and B, n = 3). WT and Lsp1–/– mice were also reconstituted with WT and Lsp1–/– leukocytes, and indicated as WTWT and Lsp1–/–Lsp1–/–, respectively (C and D, n = 34). Leukocyte recruitment was induced by intrascrotal injection of TNF (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 WTWT mice at 4 h.
In a second series of experiments, we tested responses to KC in the chimeric mice. Both sets of chimeric mice demonstrated similar responsiveness in the leukocyte rolling flux (not depicted), rolling velocity (not depicted), and adhesion (Fig. 8 A) upon placement of the KC-containing gel onto the muscle microvasculature. Again, chimeric mice that lacked LSP1 only in their leukocytes emigrated as effectively across the vasculature as WT mice (Fig. 8 B) in response to KC administration. In contrast, WT leukocytes reconstituted in Lsp1–/– mice (i.e., lacking LSP1 in endothelium) did not display significant transendothelial migration (Fig. 8 B).
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 WTLsp1–/– (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.
LSP1 is important in histamine-stimulated permeability increases in postcapillary venules
This is the first demonstration of LSP1 in endothelium and, more importantly, the first demonstration of a functional role for endothelial LSP1 in regulating leukocyte emigration. Although the mechanism by which Lsp1–/– endothelium restricts leukocyte recruitment is unclear, it is clear that LSP1 is an F-actin binding protein and involved in cytoskeletal changes in leukocytes (7, 8, 19). A very likely possibility is that the Lsp1–/– endothelium did not actively retract to permit leukocyte transendothelial migration. Therefore, we measured permeability responses in the postcapillary venules in the control WT and Lsp1–/– mice. We chose histamine, a stimulus that did not induce neutrophil emigration per se, to avoid complications associated with neutrophils emigrating in WT but not in Lsp1–/– mice. In each case, more FITC-albumin leaked into the interstitium in WT than in Lsp1–/– mice. Fig. 9 shows that at 1, 10, 30, and 60 min after 0.1 mM histamine superfusion, the permeability index in venules of WT mice was significantly higher than in venules of Lsp1–/– mice (P < 0.05). Thus, the results indicated that LSP1 has a functional role in regulating the stimulated permeability changes in postcapillary venules.
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.
LSP1 deficiency does not decrease all forms of leukocyte recruitment
When we administered IL-1? to the peritoneal cavities of WT and Lsp1–/– mice, there was very significant neutrophil recruitment in both strains of mice. In fact, the total leukocytes recovered from the peritoneal lavage were slightly higher in Lsp1–/– mice than in WT mice (at 4 h, 11.1 x 106 ± 1.2 x 106 cells in Lsp1–/– mice vs. 8.0 x 106 ± 0.7 x 106 cells in WT mice; P < 0.05), consistent with previously published results (18).
Discussion
In this paper, we have demonstrated that LSP1 appears to be an essential intracellular molecule involved in the crucial event of transendothelial migration that permits leukocytes to be recruited to sites of inflammation. Neutrophil transendothelial migration across postcapillary venules was clearly impaired in vivo in Lsp1–/– mice when compared with WT littermates. Not all leukocyte functions were inhibited inasmuch as rolling and adhesion within the vasculature (selectin- and integrin-dependent events, respectively) were not altered in Lsp1–/– mice. The impaired recruitment appeared to occur regardless of whether an exogenous chemokine was introduced or whether endogenous chemokines were produced by the local administration of TNF or IL-1?. Until now, LSP1 has been considered to be leukocyte specific; however, we report herein that this F-actin binding protein is also located in microvascular endothelium and functions to permit neutrophil transendothelial migration, suggesting for the first time both a new cellular source of LSP1 and a new critical functional role for LSP1 in endothelium.
Although previous findings of altered neutrophil recruitment in Lsp1–/– mice were considered to exclusively reflect an alteration in neutrophil function per se (18–20), in this paper, we provide evidence that endothelial LSP1 also contributes to the neutrophil recruitment. First, we demonstrated that LSP1 was located in endothelium. The protein was the same size as leukocyte LSP1 and was absent in Lsp1–/– mice. Second, chimeric mice were generated that had WT leukocytes and Lsp1–/– endothelium to delineate the importance of endothelial LSP1. The results clearly demonstrated that WT neutrophils had a profound inability to transmigrate across Lsp1–/– endothelium. In contrast, Lsp1–/– neutrophils migrated across WT endothelium with no impairment. In addition, we report that Lsp1–/– endothelium was less responsive to histamine, a molecule known to activate the endothelial cytoskeleton and induce endothelial retraction.
There is a growing body of evidence that endothelial cells actively contribute to leukocyte transendothelial migration, not only by presenting adhesion molecules including PECAM-1, CD99, and JAM-1, but also by actively retracting and forming gaps upon leukocyte adhesion to the endothelial cells and providing leukocytes a passage of lesser resistance (21, 22, 29, 34, 35). Endothelial cell to cell adherens junctions contain VE-cadherin, which links to different intracellular proteins including ?-catenin, -catenin (plakoglobin), and p120; the former two connect to actin cytoskeleton through the binding to -catenin (36, 37). The retraction process has been shown in vitro and in vivo to involve cytoskeletal rearrangement within the endothelium, leading to the disengagement of catenins and VE-cadherins and to the increase in endothelial permeability (38, 39); this retraction can be triggered by both inflammatory mediators, such as histamine, as well as by the direct process of leukocyte adhesion (21, 22, 29, 34–37, 40). Disruption of endothelial microfilaments significantly reduced leukocyte transmigration (41, 42). Although the signaling events leading to the retraction of the endothelium upstream of the cytoskeleton are not entirely clear, the prevailing view at this stage is that leukocytes engage surface molecules on the endothelium that activate intracellular signaling events, including increased intracellular Ca2+ and activation of PKC and/or myosin light chain kinase, leading to active retraction of endothelium (21, 29, 43, 44). Because LSP1 has actin-binding sites (8, 9), has been shown to play a role in adhesion-dependent polarization (19), and is associated with the cytoplasmic face of the plasma membrane (6), we propose that disruption of LSP1 perturbs the ability of endothelium to retract and, thus, decreases the histamine-induced permeability response of the endothelium and neutrophil extravasation observed in this paper. However, whether LSP1 simply functions upstream of the cytoskeletal changes or physically binds cytoskeleton remains unclear from our immunofluorescence data. It should be noted that overexpression of LSP1 in endothelium (via transfection) revealed significant cytoskeletal association (unpublished data).
It is well appreciated that, in leukocytes, LSP1 is one of several key substrates of MAPKAP kinase-2 and the latter is the direct target of p38 MAPK (10, 45). Several in vitro studies have revealed that both p38 MAPK and MAPKAP kinase-2 are important intracellular signaling molecules in the induction of chemotaxis (11–13). Recently, we have reported that inhibition of p38 MAPK in vivo resulted in both an impairment in transendothelial migration and neutrophil chemotaxis in response to the chemokine KC (13). In contrast, inhibition of p38 MAPK in vitro in human endothelium did not impair recruitment in response to the cytokine TNF (46). In this paper, lack of LSP1 caused impaired emigration in response to both TNF and KC. This clearly suggests that the LSP1 deficiency extends well beyond the role of substrate for p38 MAPK. Indeed, it has been reported that LSP1 is also a major substrate for PKC (15, 16), a molecule known to regulate endothelial retraction. An alternative explanation is that the p38 MAPK inhibitors were less effective than LSP1 deficiency. The p38 MAPK inhibitors used previously in this regard are known to specifically inhibit the p38 and p38? isoform, but not the p38 and p38 isoforms. Because endothelium can express p38, ?, , and (45), if LSP1 is downstream of each of these p38 isoforms, then this may explain the additional inhibition seen in Lsp1–/– mice versus p38 inhibitors.
The nonlethal phenotype of Lsp1–/– mice permitted manipulations that resolved the importance of this molecule in different cell types. Indeed, if the LSP1 pathway is critical within neutrophils for transendothelial migration, then, removal of LSP1 only in leukocytes by generating LSP1 chimeric mice should have still resulted in an inability of Lsp1–/– neutrophils to emigrate out of WT vasculature. Surprisingly, BM transplantation of Lsp1–/– leukocytes into WT mice restored normal transendothelial migration, suggesting that the lack of neutrophil LSP1 was not responsible for the impairment of transendothelial migration in Lsp1–/– mice. Alternative explanations included the possibilities that the irradiation process in some nonspecific manner altered the transendothelial migration phenotype and that the irradiated mice now had an enhanced responsiveness to chemokines. However, our data from the reversed BM chimeras, in which WT neutrophils failed to emigrate out of Lsp1–/– vasculature, refute these alternative explanations and support the view that neutrophil transendothelial migration is dependent on a nonleukocyte source of LSP1.
The impaired neutrophil recruitment due to LSP1 deficiency in our study in muscle appeared to be either stimulus or site specific as the work by Jongstra-Bilen and colleagues clearly revealed the opposite response (i.e., enhanced recruitment of leukocytes into the peritoneal cavity in response to thioglycolate; reference 18). We found that, whereas the recruitment of Lsp1–/– neutrophils was decreased in cremaster muscle upon IL-1? local administration, the recruitment of Lsp1–/– leukocytes into peritoneal cavity was not impaired (but increased) after IL-1? i.p. injection. Although Lsp1–/– neutrophils emigrated less into muscle in response to TNF, we also found that there was no apparent difference in the recruitment of Lsp1–/– and WT leukocytes into peritoneal cavity upon TNF i.p. administration (unpublished data). These results argue against stimulus specificity and support the notion that structural or physiological differences of the organ microvasculatures dictate organ-specific mechanisms of neutrophil recruitment (47).
This is the first documentation of a functional role for LSP1 in endothelium. Some LSP1 could be detected associated with the cytoskeleton; however, the amount was quite low. It may be that the protein only binds the cytoskeleton after leukocyte binding to the endothelium, or perhaps under flow conditions when the endothelium is under constant state of shear force. In addition, under stimulating conditions with a permeabilizing agent, perhaps the movement of LSP1 to cytoskeleton might be observed. Future studies will need to examine under what conditions and how LSP1 interacts with the actin cytoskeleton in endothelium. We will also need to determine whether the recently described function of LSP1 as a cytoskeletal ERK/MAP kinase pathway targeting protein (48) plays a role in its function as an endothelial gatekeeper of leukocyte transendothelial migration.
Materials and Methods
Animals
129/SvJ WT mice were purchased from The Jackson Laboratory. Lsp1–/– mice on the 129/SvJ background were generated by homologous recombination by Jongstra-Bilen and colleagues as described previously (18) and transferred to the University of Calgary Health Sciences Centre. Mice of these two genotypes were bred in the University Animal Centre to obtain age- and sex-matched controls. The mice between 8 and 16 wk of age were used in experiments except for the mice used for isolation of mouse endothelial cells (5–7 d old). TIE2-GFP mice were also purchased from The Jackson Laboratory. All animal protocols were approved by the Animal Care Committee of the University of Calgary and met the standards of the Canadian Association of Animal Care. All animals were kept in specific pathogen-free conditions.
Two types of BM chimeric mice were generated following the standard protocols in our laboratory (33). In brief, BM was isolated from 6–8-wk-old donor mice killed by spinal cord displacement. The BM cell suspensions (8 x 106 cells) from donor Lsp1–/– and 129/SvJ WT mice were injected into the tail vein of 129/SvJ WT and Lsp1–/– mice, respectively. Before BM cell injection, recipients were irradiated with two doses of 5 Gy -ray (Gammacell, 137Cs -irradiation source) with a 3-h interval between the two irradiations. These chimeric mice were housed in specific pathogen-free facilities for 6–8 wk to allow full humoral reconstitution before use in experiments. Initial experiments confirmed that 99% of leukocytes were from donor mice (33). Two additional control groups of mice (WT into WT and Lsp1–/– into Lsp1–/–) went through an identical protocol to ensure that the transplant procedure did not cause any untoward effects.
Intravital microscopy
Male mice were anesthetized with an i.p. injection of a mixture of 10 mg/kg xylazine (Animal Health; Bayer Inc.) and 200 mg/kg ketamine hydrochloride (Rogar/STB Inc.). For all protocols, the left jugular vein was cannulated to administer additional anesthetic or drugs when necessary. The mouse cremaster muscle preparation was used to study the behavior of leukocytes in the microcirculation and adjacent connective tissue as described previously (49). In brief, an incision was made in the scrotal skin to expose the left cremaster muscle, which was then carefully dissected free of the associated fascia. The cremaster muscle was cut longitudinally with a cautery. The testicle and the epididymis were separated from the underlying muscle and were moved into the abdominal cavity. The muscle was held flat on an optically clear viewing pedestal and was secured along the edges with 4–0 suture. The exposed tissue was superfused with 37°C warmed bicarbonate-buffered saline, pH 7.4. An intravital microscope (Axiolskip; Carl Zeiss MicroImaging, Inc.) with a x25 objective lens (Weltzlar L25/0.35; E. Leitz Inc.), and a x10 eyepiece was used to examine the cremasteric microcirculation. A video camera (5100 HS; Panasonic) was used to project the images onto a monitor, and the images were recorded for playback analysis using a videocassette recorder.
Single unbranched cremasteric venules (25–40 μm in diameter) were selected, and to minimize variability, the same section of cremasteric venule was observed throughout the experiment. The number of rolling, adherent, and emigrated leukocytes was determined offline during video playback analysis. Rolling leukocytes were defined as those cells moving at a velocity less than that of erythrocytes within a given vessel. The flux of rolling cells was measured as the number of rolling leukocytes passing by a given point in the venule per minute. Leukocyte rolling velocity was measured for the first 20 leukocytes entering the field of view at the time of recording and calculated from the time required for a leukocyte to roll along a 100-μm length of venule. A leukocyte was considered to be adherent if it remained stationary for at least 30 s, and total leukocyte adhesion was quantified as the number of adherent cells within a 100-μm length of venule in 5 min. Leukocyte emigration was defined as the number of cells in the extravascular space within a 200 x 300-μm area (0.06 mm2), adjacent to the observed venule. More than 90% of these emigrated cells were neutrophils (13, 31). Only cells adjacent to and clearly outside the vessel under study were counted as emigrated.
Induction of leukocyte recruitment in cremaster muscle
To determine whether emigration was impaired in response to cytokines, recombinant mouse TNF (0.5 or 0.1 μg; R&D Systems) or IL-1? (12.5 ng; BD Biosciences) in 200 μl of saline was injected intrascrotally into Lsp1–/–, 129/SvJ WT, or chimeric mice. The leukocyte rolling flux, rolling velocity, adherence, and emigration were measured in the cremasteric venule at 3.5, 4, and 4.5 h after the injection.
To induce neutrophil recruitment independent of cytokines, an agarose gel containing KC (CXCL1; R&D Systems) was used (13, 50). The agarose gel was prepared by adding 10 ml of 2 x HBSS to a boiling concentrated agarose solution (4% in 10 ml of distilled water). A 100-μl aliquot of this solution was removed, and KC was added to this aliquot and mixed to achieve a final concentration of 0.5 μM. To enable visualization of the gel on the cremaster muscle, a small amount of India ink was added to each preparation. A 1-mm3 piece of the mixture (KC-containing gel) was punched out using the tip of a Pasteur pipette. This piece of KC-containing gel was carefully placed on the surface of the cremaster in a preselected area 350 μm (two monitor screens wide) from a postcapillary venule. The gel was held in place using a 22 x 22-mm glass coverslip, and the tissue was superfused beneath the coverslip at a slow rate (0.35 ml/min) to create a chemotactic gradient that permitted emigration. The image was recorded for 90 min: 30 min for control without gel and 60 min with KC-containing gel. In control experiments, only the gel (without KC addition) was placed on the surface of the cremaster muscle.
Microvascular permeability measurement
The degree of vascular albumin leakage from cremasteric venules of Lsp1–/– and control 129/SvJ mice was quantified as described previously (51). In brief, 25 mg/kg FITC-labeled BSA (Sigma-Aldrich) was administered to the mice i.v. at the start of the experiment, and FITC-derived fluorescence (excitation wavelength, 450–490 nm; emission wavelength, 520 nm) was detected using a silicon-intensified charge-coupled device camera (model C-2400-80; Hamamatsu Photonics). Image analysis software (Optimas; Bioscan Inc.) was used to determine the intensity of FITC-albumin–derived fluorescence within the lumen of the venule and in the adjacent perivascular tissue. Background was defined as the fluorescence intensity before FITC-albumin administration. The exposed cremaster muscle was superfused with 0.1 mM histamine dihydrochloride (Sigma-Aldrich) in 37°C warmed bicarbonate-buffered saline. The index of vascular albumin leakage (permeability index) at different time points after histamine superfusion was determined according to the following ratio expressed as a percentage: (mean interstitial intensity – background)/(venular intensity – background) (51).
Harvesting endothelial cells and leukocytes
Acute mouse peritonitis was induced to obtain emigrated leukocytes from Lsp1–/– and control 129/SvJ WT mice. 3 h after an i.p. injection of 1% oyster glycogen (in 1 ml saline; Sigma-Aldrich), leukocytes were lavaged from the peritoneum and prepared for Western blotting (see the next paragraph). Mouse primary lung endothelial cells were isolated from 5–7-d-old Lsp1–/– and control 129/SvJ WT mice, and were cultured according to the protocols described previously (52). Using this protocol with TIE2-GFP mice and flow cytometry, we verified that 93–98% of the isolated cells were GFP positive, confirming that the majority of the purified cells were of endothelial cell origin (53). Freshly isolated mouse endothelial cells were cultured in microvascular endothelial cell medium-2 (Clonetics EGM-2MV BulletKit; Cambrex Bio Science) in 35-mm Petri dishes precoated with 20 μg/ml mouse laminin (Upstate Biotechnology). After reaching confluence in 5–6 d, the cells were either used for Western blotting or trypsinized and subcultured on laminin-coated 22 x 22-mm glass coverslips (at 30,000 cells/coverslip) contained in 35-mm Petri dishes.
Western blot and RT-PCR analysis
The polyclonal anti-LSP1 serum was made in rabbits against mouse recombinant LSP1 protein (6). Although, in leukocytes, the anti-LSP1 serum detected a single band at the appropriate size for LSP1, a second band of 78 kD was detected in endothelium. To remove this reactivity, the GST-LSP1 fusion proteins containing LSP1 residues 1–178 or 179–330 were constructed, subcloned, and expressed in Escherichia coli BL21 (DE3) cells as described previously (54). These two fusion proteins were allowed to conjugate agarose-glutathione beads and were packed into separate glass columns. The polyclonal anti-LSP1 serum was flowed at a rate of <0.15 ml/min (at 4°C) through the different columns containing at least 10-fold molar excess fusion proteins (residues 1–178 or 179–330). The flow-through fractions containing >1.5 mg/ml protein concentrations, which were the anti–COOH-terminal LSP1 and anti–NH2-terminal LSP1, respectively, were collected, pooled separately, and used in Western blotting assays and immunofluorescence microscopy. As a control, the anti-LSP1 serum was also absorbed against both GST-LSP1 fusion proteins and used in Western blotting.
Freshly isolated mouse leukocytes and mouse primary lung endothelial cells were used for Western blot and RT-PCR analysis. Whole cell lysates were prepared from these cells using Laemmli buffer with 10% ?-mercaptoethanol, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. The proteins were separated by electrophoresis in 10% SDS-polyacrylamide gels, transferred to a PVDF Hybond-P transfer membrane (Amersham Biosciences), and blotted using a specific polyclonal rabbit anti–mouse LSP1 serum (at 1:2,000 dilution) as described previously (6) or the anti–COOH-terminal LSP1 and anti–NH2-terminal LSP1 as described before. After washing, the membrane was incubated with a secondary, horseradish peroxidase–conjugated goat anti–rabbit IgG and treated with enhanced chemiluminescence reagents (ECL kit; Amersham Biosciences). The blotted bands were detected with high performance autoradiography films from Amersham Biosciences. RT-PCR was performed using total RNA (100 ng for each cell type) extracted from freshly isolated leukocytes, mouse primary lung endothelial cells, and LSP1 primer pair A1/A4 as described previously (4). The PCR products were electrophoresed by agarose gel, stained with ethidium bromide, and analyzed by high sensitivity Fluor-S Multimager MAX scanner (Bio-Rad Laboratories) upon dark subtraction.
Immunofluorescence microscopy
All rinsing, incubation, and dilution of antibodies was performed in basal buffer that contained 137 NaCl, 5 KCl, 1.1 Na2HPO4, 0.4 KH2PO4, 4 NaHCO3, 5.5 glucose, 4.15 PIPES disodium salt, 2 EGTA, and 4.15 MgCl2 in mM, pH 7.2, at room temperature. Mouse lung primary endothelial cells grown on glass coverslips for 24 h were fixed with 4% formalin, permeabilized with 0.1% Triton X-100, and incubated with 10 μg/ml glycine. Primary antibodies used were the anti–COOH-terminal LSP1 and anti–NH2-terminal LSP1 as described before for 30 min where the protein concentrations were both 0.4 mg/ml in the two blotting solutions, or the original rabbit polyclonal anti-LSP1 serum (at 1:100 dilution). After rinsing three times with 0.1% Tween-20, the coverslips were incubated with Cy3-conjugated goat anti–rabbit IgG for 30 min. After rinsing with basal buffer, the coverslips were mounted in 90% glycerol. Observations were performed on an Olympus IX-70 fluorescence microscope (Olympus). Fluorescence images were captured using OpenLab software (version 3.1.5; Improvision Inc.).
To determine whether human endothelial cells express LSP1, we isolated and cultured HUVECs as described previously (46). Once the HUVECs were confluent, they were passaged onto fibronectin-coated glass coverslips. After culture for 24–48 h, these HUVECs were stained as outlined before with 2.5 μg/ml mouse anti–human LSP1 mAb (clone 16; BD Biosciences) and goat anti–mouse IgG conjugated with Texas Red (Molecular Probes). DAPI (4',6-diamidino-2-phenylindole, dihydrochloride; Molecular Probes) was used for nuclear staining. To confirm that endothelial cells and not a contaminating cell type expressed LSP1, we dual labeled the HUVECs with FITC-conjugated anti–human VE-cadherin (Bender Medsystems) in combination with LSP1 staining. To assess the association of LSP1 with the cytoskeleton, we labeled HUVECs with anti-LSP1 and phalloidin (Alexa Fluor conjugated; Molecular Probes).
Statistical analysis
The data are expressed as means ± SEM. A Student's t test was applied to compare the statistical difference within two groups, and analysis of variance was used for the comparison of the differences in more than two groups. A p-value of <0.05 was considered statistically significant.
Acknowledgments
We thank L. Zbytnuik and K. Jorgensen for their expert assistance in animal care.
This work was supported in part by a Canadian Institutes of Health Research group grant and by the Heart and Stroke Foundation of Canada. L. Liu is supported by a fellowship from Heart and Stroke Foundation of Canada and Alberta Heritage Foundation for Medical Research. D.C. Cara has a postdoctoral fellowship from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq, Brasilia, Brazil. P. Kubes is an Alberta Heritage Foundation for Medical Research Scientist and a Canada Research Chair.
References
Jongstra, J., G.F. Tidmarsh, J. Jongstra-Bilen, and M.M. Davis. 1988. A new lymphocyte-specific gene which encodes a putative Ca2+-binding protein is not expressed in transformed T lymphocyte lines. J. Immunol. 141:3999–4004.
Jongstra-Bilen, J., A.J. Young, R. Chong, and J. Jongstra. 1990. Human and mouse LSP1 genes code for highly conserved phosphoproteins. J. Immunol. 144:1104–1110.
Pulford, K., M. Jones, A.H. Banham, E. Haralambieva, and D.Y. Mason. 1999. Lymphocyte-specific protein 1: a specific marker of human leucocytes. Immunology. 96:262–271.
Jongstra, J., M.E. Ittel, N.N. Iscove, and G. Brady. 1994. The LSP1 gene is expressed in cultured normal and transformed mouse macrophages. Mol. Immunol. 31:1125–1131.
Li, Y., A. Guerrero, and T.H. Howard. 1995. The actin-binding protein, lymphocyte-specific protein 1, is expressed in human leukocytes and human myeloid and lymphoid cell lines. J. Immunol. 155:3563–3569.
Klein, D.P., J. Jongstra-Bilen, K. Ogryzlo, R. Chong, and J. Jongstra. 1989. Lymphocyte-specific Ca2+-binding protein LSP1 is associated with the cytoplasmic face of the plasma membrane. Mol. Cell. Biol. 9:3043–3048.
Klein, D.P., S. Galea, and J. Jongstra. 1990. The lymphocyte-specific protein LSP1 is associated with the cytoskeleton and co-caps with membrane IgM. J. Immunol. 145:2967–2973.
Jongstra-Bilen, J., P.A. Janmey, J.H. Hartwig, S. Galea, and J. Jongstra. 1992. The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain. J. Cell Biol. 118:1443–1453.
Zhang, Q., Y. Li, and T.H. Howard. 2000. Human lymphocyte-specific protein 1, the protein overexpressed in neutrophil actin dysfunction with 47-kDa and 89-kDa protein abnormalities (NAD 47/89), has multiple F-actin binding domains. J. Immunol. 165:2052–2058.
Huang, C.K., L. Zhan, Y. Ai, and J. Jongstra. 1997. LSP1 is the major substrate for mitogen-activated protein kinase-activated protein kinase 2 in human neutrophils. J. Biol. Chem. 272:17–19.
Hannigan, M.O., L. Zhan, Y. Ai, A. Kotlyarov, M. Gaestel, and C.K. Huang. 2001. Abnormal migration phenotype of mitogen-activated protein kinase-activated protein kinase 2–/– neutrophils in Zigmond chambers containing formyl-methionyl-leucyl-phenylalanine gradients. J. Immunol. 167:3953–3961.
Zu, Y.L., J. Qi, A. Gilchrist, G.A. Fernandez, D. Vazquez-Abad, D.L. Kreutzer, C.K. Huang, and R.I. Sha'afi. 1998. p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF- or FMLP stimulation. J. Immunol. 160:1982–1989.
Cara, D.C., J. Kaur, M. Forster, D.M. McCafferty, and P. Kubes. 2001. Role of p38 mitogen-activated protein kinase in chemokine-induced emigration and chemotaxis in vivo. J. Immunol. 167:6552–6558.
Stokoe, D., K. Engel, D.G. Campbell, P. Cohen, and M. Gaestel. 1992. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett. 313:307–313.
Carballo, E., D. Colomer, J.L. Vives-Corrons, P.J. Blackshear, and J. Gil. 1996. Characterization and purification of a protein kinase C substrate in human B cells. Identification as lymphocyte-specific protein 1 (LSP1). J. Immunol. 156:1709–1713.
Matsumoto, N., S. Kojima, T. Osawa, and S. Toyoshima. 1995. Protein kinase C phosphorylates p50 LSP1 and induces translocation of p50 LSP1 in T lymphocytes. J. Biochem. (Tokyo). 117:222–229.
Laudanna, C., D. Mochly-Rosen, T. Liron, G. Constantin, and E.C. Butcher. 1998. Evidence of protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis. J. Biol. Chem. 273:30306–30315.
Jongstra-Bilen, J., V.L. Misener, C. Wang, H. Ginzberg, A. Auerbach, A.L. Joyner, G.P. Downey, and J. Jongstra. 2000. LSP1 modulates leukocyte populations in resting and inflamed peritoneum. Blood. 96:1827–1835.
Wang, C., H. Hayashi, R. Harrison, B. Chiu, J.R. Chan, H.L. Ostergaard, R.D. Inman, J. Jongstra, M.I. Cybulsky, and J. Jongstra-Bilen. 2002. Modulation of Mac-1 (CD11b/CD18)-mediated adhesion by the leukocyte-specific protein 1 is key to its role in neutrophil polarization and chemotaxis. J. Immunol. 169:415–423.
Hannigan, M., L. Zhan, Y. Ai, and C.K. Huang. 2001. Leukocyte-specific gene 1 protein (LSP1) is involved in chemokine KC-activated cytoskeletal reorganization in murine neutrophils in vitro. J. Leukoc. Biol. 69:497–504.
Luscinskas, F.W., S. Ma, A. Nusrat, C.A. Parkos, and S.K. Shaw. 2002. Leukocyte transendothelial migration: a junctional affair. Semin. Immunol. 14:105–113.
Vestweber, D. 2002. Regulation of endothelial cell contacts during leukocyte extravasation. Curr. Opin. Cell Biol. 14:587–593.
Kayyali, U.S., C.M. Pennella, C. Trujillo, O. Villa, M. Gaestel, and P.M. Hassoun. 2002. Cytoskeletal changes in hypoxic pulmonary endothelial cells are dependent on MAPK-activated protein kinase MK2. J. Biol. Chem. 277:42596–42602.
Park, J.H., N. Okayama, D. Gute, A. Krsmanovic, H. Battarbee, and J.S. Alexander. 1999. Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton. Am. J. Physiol. 277:C1066–C1074.
Huot, J., F. Houle, F. Marceau, and J. Landry. 1997. Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ. Res. 80:383–392.
Nwariaku, F.E., J. Chang, X. Zhu, Z. Liu, S.L. Duffy, N.H. Halaihel, L. Terada, and R.H. Turnage. 2002. The role of p38 MAP kinase in tumor necrosis factor-induced redistribution of vascular endothelial cadherin and increased endothelial permeability. Shock. 18:82–85.
Laird, S.M., A. Graham, A. Paul, G.W. Gould, C. Kennedy, and R. Plevin. 1998. Tumour necrosis factor stimulates stress-activated protein kinases and the inhibition of DNA synthesis in cultures of bovine aortic endothelial cells. Cell. Signal. 10:473–480.
Rousseau, S., F. Houle, J. Landry, and J. Huot. 1997. p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene. 15:2169–2177.
Huang, A.J., J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells. J. Cell Biol. 120:1371–1380.
Garcia, J.G., A.D. Verin, M. Herenyiova, and D. English. 1998. Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration. J. Appl. Physiol. 84:1817–1821.
Thompson, R.D., K.E. Noble, K.Y. Larbi, A. Dewar, G.S. Duncan, T.W. Mak, and S. Nourshargh. 2001. Platelet-endothelial cell adhesion molecule-1 (PECAM-1)–deficient mice demonstrate a transient and cytokine-specific role for PECAM-1 in leukocyte migration through the perivascular basement membrane. Blood. 97:1854–1860.
Young, R.E., R.D. Thompson, and S. Nourshargh. 2002. Divergent mechanisms of action of the inflammatory cytokines interleukin 1-? and tumour necrosis factor- in mouse cremasteric venules. Br. J. Pharmacol. 137:1237–1246.
Carvalho-Tavares, J., M.J. Hickey, J. Hutchison, J. Michaud, I.T. Sutcliffe, and P. Kubes. 2000. A role for platelets and endothelial selectins in tumor necrosis factor--induced leukocyte recruitment in the brain microvasculature. Circ. Res. 87:1141–1148.
Del Maschio, A., A. Zanetti, M. Corada, Y. Rival, L. Ruco, M.G. Lampugnani, and E. Dejana. 1996. Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions. J. Cell Biol. 135:497–510.
Johnson-Leger, C., M. Aurrand-Lions, and B.A. Imhof. 2000. The parting of the endothelium: miracle, or simply a junctional affair? J. Cell Sci. 113:921–933.
Dejana, E. 1996. Endothelial adherens junctions: implications in the control of vascular permeability and angiogenesis. J. Clin. Invest. 98:1949–1953.
Dejana, E., R. Spagnuolo, and G. Bazzoni. 2001. Interendothelial junctions and their role in the control of angiogenesis, vascular permeability and leukocyte transmigration. Thromb. Haemost. 86:308–315.
Waschke, J., W. Baumgartner, R.H. Adamson, M. Zeng, K. Aktories, H. Barth, C. Wilde, F.E. Curry, and D. Drenckhahn. 2004. Requirement of Rac activity for maintenance of capillary endothelial barrier properties. Am. J. Physiol. Heart Circ. Physiol. 286:H394–H401.
Adamson, R.H., F.E. Curry, G. Adamson, B. Liu, Y. Jiang, K. Aktories, H. Barth, A. Daigeler, N. Golenhofen, W. Ness, and D. Drenckhahn. 2002. Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice. J. Physiol. 539:295–308.
Shaw, S.K., P.S. Bamba, B.N. Perkins, and F.W. Luscinskas. 2001. Real-time imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium. J. Immunol. 167:2323–2330.
Kielbassa, K., C. Schmitz, and V. Gerke. 1998. Disruption of endothelial microfilaments selectively reduces the transendothelial migration of monocytes. Exp. Cell Res. 243:129–141.
Hordijk, P.L., E. Anthony, F.P. Mul, R. Rientsma, L.C. Oomen, and D. Roos. 1999. Vascular-endothelial-cadherin modulates endothelial monolayer permeability. J. Cell Sci. 112:1915–1923.
Su, W.H., H.I. Chen, J.P. Huang, and C.J. Jen. 2000. Endothelial i signaling during transmigration of polymorphonuclear leukocytes. Blood. 96:3816–3822.
Lum, H., and A.B. Malik. 1994. Regulation of vascular endothelial barrier function. Am. J. Physiol. 267:L223–L241.
Herlaar, E., and Z. Brown. 1999. p38 MAPK signalling cascades in inflammatory disease. Mol. Med. Today. 5:439–447.
Kaur, J., R.C. Woodman, and P. Kubes. 2003. P38 MAPK: critical molecule in thrombin-induced NF-B-dependent leukocyte recruitment. Am. J. Physiol. Heart Circ. Physiol. 284:H1095–H1103.
Liu, L., and P. Kubes. 2003. Molecular mechanisms of leukocyte recruitment: organ-specific mechanisms of action. Thromb. Haemost. 89:213–220.
Harrison, R.E., B.A. Sikorski, and J. Jongstra. 2004. Leukocyte-specific protein 1 targets the ERK/MAP kinase scaffold protein KSR and MEK1 and ERK2 to the actin cytoskeleton. J. Cell Sci. 117:2151–2157.
Kanwar, S., D.C. Bullard, M.J. Hickey, C.W. Smith, A.L. Beaudet, B.A. Wolitzky, and P. Kubes. 1997. The association between 4-integrin, P-selectin, and E-selectin in an allergic model of inflammation. J. Exp. Med. 185:1077–1087.
Hickey, M.J., M. Forster, D. Mitchell, J. Kaur, C. De Caigny, and P. Kubes. 2000. L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo. J. Immunol. 165:7164–7170.
Kurose, I., P. Kubes, R. Wolf, D.C. Anderson, J. Paulson, M. Miyasaka, and D.N. Granger. 1993. Inhibition of nitric oxide production. Mechanisms of vascular albumin leakage. Circ. Res. 73:164–171.
Bowden, R.A., Z.M. Ding, E.M. Donnachie, T.K. Petersen, L.H. Michael, C.M. Ballantyne, and A.R. Burns. 2002. Role of 4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium. Circ. Res. 90:562–569.
Motoike, T., S. Loughna, E. Perens, B.L. Roman, W. Liao, T.C. Chau, C.D. Richardson, T. Kawate, J. Kuno, B.M. Weinstein, et al. 2000. Universal GFP reporter for the study of vascular development. Genesis. 28:75–81.
Wong, M.J., I.A. Malapitan, B.A. Sikorski, and J. Jongstra. 2003. A cell-free binding assay maps the LSP1 cytoskeletal binding site to the COOH-terminal 30 amino acids. Biochim. Biophys. Acta. 1642:17–24.(Lixin Liu1, Denise C. Car)