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L-selectin-mediated leukocyte adhesion in vivo: microvillous distribution determines tethering efficiency, but not rolling velocity.

Stein JV, Cheng G, Stockton BM, Fors BP, Butcher EC, von Andrian UH - J. Exp. Med. (1999)

Bottom Line: In the narrow venules, tethering of cells with cell body expression may have been aided by forced margination through collision with erythrocytes.L-selectin transfected cells rolled 10-fold faster than E-selectin transfectants.Interestingly, rolling velocity histograms of cell lines expressing equivalent copy numbers of the same ectodomain were always similar, irrespective of the topographic distribution.

View Article: PubMed Central - PubMed

Affiliation: Center for Blood Research and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Adhesion receptors that are known to initiate contact (tethering) between blood-borne leukocytes and their endothelial counterreceptors are frequently concentrated on the microvilli of leukocytes. Other adhesion molecules are displayed either randomly or preferentially on the planar cell body. To determine whether ultrastructural distribution plays a role during tethering in vivo, we used pre-B cell transfectants expressing L- or E-selectin ectodomains linked to transmembrane/intracellular domains that mediated different surface distribution patterns. We analyzed the frequency and velocity of transfectant rolling in high endothelial venules of peripheral lymph nodes using an intravital microscopy model. Ectodomains on microvilli conferred a higher efficiency at initiating rolling than random distribution which, in turn, was more efficient than preferential expression on the cell body. The role of microvillous presentation was less accentuated in venules below 20 micrometers in diameter than in larger venules. In the narrow venules, tethering of cells with cell body expression may have been aided by forced margination through collision with erythrocytes. L-selectin transfected cells rolled 10-fold faster than E-selectin transfectants. Interestingly, rolling velocity histograms of cell lines expressing equivalent copy numbers of the same ectodomain were always similar, irrespective of the topographic distribution. Our data indicate that the distribution of adhesion receptors has a dramatic impact on contact initiation between leukocytes and endothelial cells, but does not play a role once rolling has been established.

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Rolling fractions of L1-2 transfectants expressing the L-selectin ED in PLN venules. L1-2  cells transfected with wild-type or chimeric L-selectin were fluorescently labeled with BCECF and  observed in PLN venules of different order. The percentage of rolling cells in the total cell flux (rolling fraction) was determined in individual venules for each pair of transfectants, as described in Materials and Methods. Rolling fractions of each member of a pair that were determined in the same  venule are connected by straight lines. All transfectants showed highest rolling fractions in HEV of  orders IV and V, gradually decreasing when cells entered lower order venules. Separate experiments  were performed to compare rolling fractions between (A) wild-type L-selectin and L/CD31; (B)  wild-type L-selectin and L/CD44; and (C) L/CD31 and L/CD44. (D) Relative rolling fraction of  L1-2 transfectants in all venules after normalizing the transfectant with higher rolling fraction to 100%  in each venule; data are shown as mean ± SEM of rolling fractions in 21–36 venules from three to six  independent experiments.
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Figure 2: Rolling fractions of L1-2 transfectants expressing the L-selectin ED in PLN venules. L1-2 cells transfected with wild-type or chimeric L-selectin were fluorescently labeled with BCECF and observed in PLN venules of different order. The percentage of rolling cells in the total cell flux (rolling fraction) was determined in individual venules for each pair of transfectants, as described in Materials and Methods. Rolling fractions of each member of a pair that were determined in the same venule are connected by straight lines. All transfectants showed highest rolling fractions in HEV of orders IV and V, gradually decreasing when cells entered lower order venules. Separate experiments were performed to compare rolling fractions between (A) wild-type L-selectin and L/CD31; (B) wild-type L-selectin and L/CD44; and (C) L/CD31 and L/CD44. (D) Relative rolling fraction of L1-2 transfectants in all venules after normalizing the transfectant with higher rolling fraction to 100% in each venule; data are shown as mean ± SEM of rolling fractions in 21–36 venules from three to six independent experiments.

Mentions: BCECF-labeled transfectants were injected through the femoral artery and observed in subiliac PLN microvessels. These vessels are classified in up to five branching orders in a typical nodal venular tree as described previously (3, 5). In agreement with previous studies of L1-2 transfectants expressing wild-type L-selectin, we found that their rolling fraction was highest in immediate postcapillary venules (orders IV and V), gradually decreasing in successively larger venules downstream (orders III to I) (5). This decrease in rolling fractions was shown to correlate with a decrease in PNAd density (29). Accordingly, we found a similar spatial preference in rolling for chimeric L-selectin transfectants (Fig. 2, A–C); irrespective of the ultrastructural distribution of L-selectin EDs, all cell lines rolled consistently more frequently in high order venules where L-selectin ligands were abundant, and displayed much less rolling in medullary collecting venules with sparse PNAd expression. Tethering and rolling was mediated by the EDs of transfected selectins as mock-transfected L1-2 cells were not able to tether and roll in this model (5). In agreement with previous studies with L1-2 transfectants in murine PLN, we did not observe transfectants that became stuck (stationary for ≥30 s) in any postcapillary venule analyzed, probably because these cells express very few β2 integrins which are necessary for firm adhesion in PLN HEV (3, 5).


L-selectin-mediated leukocyte adhesion in vivo: microvillous distribution determines tethering efficiency, but not rolling velocity.

Stein JV, Cheng G, Stockton BM, Fors BP, Butcher EC, von Andrian UH - J. Exp. Med. (1999)

Rolling fractions of L1-2 transfectants expressing the L-selectin ED in PLN venules. L1-2  cells transfected with wild-type or chimeric L-selectin were fluorescently labeled with BCECF and  observed in PLN venules of different order. The percentage of rolling cells in the total cell flux (rolling fraction) was determined in individual venules for each pair of transfectants, as described in Materials and Methods. Rolling fractions of each member of a pair that were determined in the same  venule are connected by straight lines. All transfectants showed highest rolling fractions in HEV of  orders IV and V, gradually decreasing when cells entered lower order venules. Separate experiments  were performed to compare rolling fractions between (A) wild-type L-selectin and L/CD31; (B)  wild-type L-selectin and L/CD44; and (C) L/CD31 and L/CD44. (D) Relative rolling fraction of  L1-2 transfectants in all venules after normalizing the transfectant with higher rolling fraction to 100%  in each venule; data are shown as mean ± SEM of rolling fractions in 21–36 venules from three to six  independent experiments.
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Related In: Results  -  Collection

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Figure 2: Rolling fractions of L1-2 transfectants expressing the L-selectin ED in PLN venules. L1-2 cells transfected with wild-type or chimeric L-selectin were fluorescently labeled with BCECF and observed in PLN venules of different order. The percentage of rolling cells in the total cell flux (rolling fraction) was determined in individual venules for each pair of transfectants, as described in Materials and Methods. Rolling fractions of each member of a pair that were determined in the same venule are connected by straight lines. All transfectants showed highest rolling fractions in HEV of orders IV and V, gradually decreasing when cells entered lower order venules. Separate experiments were performed to compare rolling fractions between (A) wild-type L-selectin and L/CD31; (B) wild-type L-selectin and L/CD44; and (C) L/CD31 and L/CD44. (D) Relative rolling fraction of L1-2 transfectants in all venules after normalizing the transfectant with higher rolling fraction to 100% in each venule; data are shown as mean ± SEM of rolling fractions in 21–36 venules from three to six independent experiments.
Mentions: BCECF-labeled transfectants were injected through the femoral artery and observed in subiliac PLN microvessels. These vessels are classified in up to five branching orders in a typical nodal venular tree as described previously (3, 5). In agreement with previous studies of L1-2 transfectants expressing wild-type L-selectin, we found that their rolling fraction was highest in immediate postcapillary venules (orders IV and V), gradually decreasing in successively larger venules downstream (orders III to I) (5). This decrease in rolling fractions was shown to correlate with a decrease in PNAd density (29). Accordingly, we found a similar spatial preference in rolling for chimeric L-selectin transfectants (Fig. 2, A–C); irrespective of the ultrastructural distribution of L-selectin EDs, all cell lines rolled consistently more frequently in high order venules where L-selectin ligands were abundant, and displayed much less rolling in medullary collecting venules with sparse PNAd expression. Tethering and rolling was mediated by the EDs of transfected selectins as mock-transfected L1-2 cells were not able to tether and roll in this model (5). In agreement with previous studies with L1-2 transfectants in murine PLN, we did not observe transfectants that became stuck (stationary for ≥30 s) in any postcapillary venule analyzed, probably because these cells express very few β2 integrins which are necessary for firm adhesion in PLN HEV (3, 5).

Bottom Line: In the narrow venules, tethering of cells with cell body expression may have been aided by forced margination through collision with erythrocytes.L-selectin transfected cells rolled 10-fold faster than E-selectin transfectants.Interestingly, rolling velocity histograms of cell lines expressing equivalent copy numbers of the same ectodomain were always similar, irrespective of the topographic distribution.

View Article: PubMed Central - PubMed

Affiliation: Center for Blood Research and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Adhesion receptors that are known to initiate contact (tethering) between blood-borne leukocytes and their endothelial counterreceptors are frequently concentrated on the microvilli of leukocytes. Other adhesion molecules are displayed either randomly or preferentially on the planar cell body. To determine whether ultrastructural distribution plays a role during tethering in vivo, we used pre-B cell transfectants expressing L- or E-selectin ectodomains linked to transmembrane/intracellular domains that mediated different surface distribution patterns. We analyzed the frequency and velocity of transfectant rolling in high endothelial venules of peripheral lymph nodes using an intravital microscopy model. Ectodomains on microvilli conferred a higher efficiency at initiating rolling than random distribution which, in turn, was more efficient than preferential expression on the cell body. The role of microvillous presentation was less accentuated in venules below 20 micrometers in diameter than in larger venules. In the narrow venules, tethering of cells with cell body expression may have been aided by forced margination through collision with erythrocytes. L-selectin transfected cells rolled 10-fold faster than E-selectin transfectants. Interestingly, rolling velocity histograms of cell lines expressing equivalent copy numbers of the same ectodomain were always similar, irrespective of the topographic distribution. Our data indicate that the distribution of adhesion receptors has a dramatic impact on contact initiation between leukocytes and endothelial cells, but does not play a role once rolling has been established.

Show MeSH
Related in: MedlinePlus