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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 velocities of L- and E-selectin  transfectants are independent of ED distribution in  PLN venules. Velocity histograms were calculated  as in Fig. 5 except that individual cells of transfectants were assigned to velocity classes with Vroll >  0 μm/s to <50 μm/s; 50 to <100 μm/s; and so  on (L-selectin transfectants) or >0 μm/s to <5  μm/s; 5 μm/s to <10 μm/s; and so on (E-selectin  transfectants). Cumulative velocity curves were calculated as in Fig. 5 B. Velocity histogram and cumulative velocity curve of (A and B) wild-type  L-selectin and L/CD31; (C and D) wild-type  L-selectin and C/CD44; (E and F) L/CD31 and  L/CD44 transfectants; and (G and H) wild-type  E-selectin and E/L. n = number of cells/venules/ experiments analyzed.
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Figure 6: Rolling velocities of L- and E-selectin transfectants are independent of ED distribution in PLN venules. Velocity histograms were calculated as in Fig. 5 except that individual cells of transfectants were assigned to velocity classes with Vroll > 0 μm/s to <50 μm/s; 50 to <100 μm/s; and so on (L-selectin transfectants) or >0 μm/s to <5 μm/s; 5 μm/s to <10 μm/s; and so on (E-selectin transfectants). Cumulative velocity curves were calculated as in Fig. 5 B. Velocity histogram and cumulative velocity curve of (A and B) wild-type L-selectin and L/CD31; (C and D) wild-type L-selectin and C/CD44; (E and F) L/CD31 and L/CD44 transfectants; and (G and H) wild-type E-selectin and E/L. n = number of cells/venules/ experiments analyzed.

Mentions: To determine the role of ED distribution on Vroll, we carried out a detailed analysis of single cell rolling velocities for each pair of transfectants. Chimeric transfectants expressing the L-selectin ectodomain showed similar average Vroll when compared to their wild-type or other chimeric counterparts (Table I). For further analysis of L1-2 cell rolling behavior in PLN HEV, velocity histograms and cumulative velocity curves were calculated as shown in Fig. 5. The velocity histograms of pairs of cell lines tested in the same preparation were comparable between all L-selectin transfectants (Fig. 6, A, C, and E). Accordingly, the cumulative velocity curves (Fig. 6, B, D, and F) were nearly superimposable. Likewise, E/L and wild-type E-selectin transfectants were found to have similar velocity histograms and cumulative velocity curves (Fig. 6, G and H). A statistical comparison of the medians of the L/CD31-L/CD44 pair, but not of any other pair, revealed a significant difference (Mann-Whitney U test; P < 0.05). The low Vroll of L/CD31 cells in this group may have been due to their unusually high expression of L-selectin EDs and relatively low shear rates (Table I). No significant differences were found in Vroll or Vrel with L/CD31 transfectants that expressed lower ED levels during comparison with wild-type L-selectin transfectants.


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 velocities of L- and E-selectin  transfectants are independent of ED distribution in  PLN venules. Velocity histograms were calculated  as in Fig. 5 except that individual cells of transfectants were assigned to velocity classes with Vroll >  0 μm/s to <50 μm/s; 50 to <100 μm/s; and so  on (L-selectin transfectants) or >0 μm/s to <5  μm/s; 5 μm/s to <10 μm/s; and so on (E-selectin  transfectants). Cumulative velocity curves were calculated as in Fig. 5 B. Velocity histogram and cumulative velocity curve of (A and B) wild-type  L-selectin and L/CD31; (C and D) wild-type  L-selectin and C/CD44; (E and F) L/CD31 and  L/CD44 transfectants; and (G and H) wild-type  E-selectin and E/L. n = number of cells/venules/ experiments analyzed.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1887701&req=5

Figure 6: Rolling velocities of L- and E-selectin transfectants are independent of ED distribution in PLN venules. Velocity histograms were calculated as in Fig. 5 except that individual cells of transfectants were assigned to velocity classes with Vroll > 0 μm/s to <50 μm/s; 50 to <100 μm/s; and so on (L-selectin transfectants) or >0 μm/s to <5 μm/s; 5 μm/s to <10 μm/s; and so on (E-selectin transfectants). Cumulative velocity curves were calculated as in Fig. 5 B. Velocity histogram and cumulative velocity curve of (A and B) wild-type L-selectin and L/CD31; (C and D) wild-type L-selectin and C/CD44; (E and F) L/CD31 and L/CD44 transfectants; and (G and H) wild-type E-selectin and E/L. n = number of cells/venules/ experiments analyzed.
Mentions: To determine the role of ED distribution on Vroll, we carried out a detailed analysis of single cell rolling velocities for each pair of transfectants. Chimeric transfectants expressing the L-selectin ectodomain showed similar average Vroll when compared to their wild-type or other chimeric counterparts (Table I). For further analysis of L1-2 cell rolling behavior in PLN HEV, velocity histograms and cumulative velocity curves were calculated as shown in Fig. 5. The velocity histograms of pairs of cell lines tested in the same preparation were comparable between all L-selectin transfectants (Fig. 6, A, C, and E). Accordingly, the cumulative velocity curves (Fig. 6, B, D, and F) were nearly superimposable. Likewise, E/L and wild-type E-selectin transfectants were found to have similar velocity histograms and cumulative velocity curves (Fig. 6, G and H). A statistical comparison of the medians of the L/CD31-L/CD44 pair, but not of any other pair, revealed a significant difference (Mann-Whitney U test; P < 0.05). The low Vroll of L/CD31 cells in this group may have been due to their unusually high expression of L-selectin EDs and relatively low shear rates (Table I). No significant differences were found in Vroll or Vrel with L/CD31 transfectants that expressed lower ED levels during comparison with wild-type L-selectin transfectants.

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