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The role of chemokines in the microenvironmental control of T versus B cell arrest in Peyer's patch high endothelial venules.

Warnock RA, Campbell JJ, Dorf ME, Matsuzawa A, McEvoy LM, Butcher EC - J. Exp. Med. (2000)

Bottom Line: We show that T cells roll on most Peyer's patch high endothelial venules (PP-HEVs), but preferentially arrest in segments displaying high levels of luminal secondary lymphoid tissue chemokine (SLC) (6Ckine, Exodus-2, thymus-derived chemotactic agent 4 [TCA-4]).Remarkably, sites of T and B cell firm adhesion are segregated in PPs, with HEVs supporting B cell accumulation concentrated in or near follicles, the target domain of most B cells entering PPs, whereas T cells preferentially accumulate in interfollicular HEVs.Our findings reveal a fundamental difference in signaling requirements for PP-HEV recognition by T and B cells, and describe an unexpected level of specialization of HEVs that may allow differential, segmental control of lymphocyte subset recruitment into functionally distinct lymphoid microenvironments in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, Department of Pathology. Stanford University Medical School, Stanford, California, USA.

ABSTRACT
Chemokines have been hypothesized to contribute to the selectivity of lymphocyte trafficking not only as chemoattractants, but also by triggering integrin-dependent sticking (arrest) of circulating lymphocytes at venular sites of extravasation. We show that T cells roll on most Peyer's patch high endothelial venules (PP-HEVs), but preferentially arrest in segments displaying high levels of luminal secondary lymphoid tissue chemokine (SLC) (6Ckine, Exodus-2, thymus-derived chemotactic agent 4 [TCA-4]). This arrest is selectively inhibited by functional deletion (desensitization) of CC chemokine receptor 7 (CCR7), the receptor for SLC and for macrophage inflammatory protein (MIP)-3beta (EBV-induced molecule 1 ligand chemokine [ELC]), and does not occur in mutant DDD/1 mice that are deficient in these CCR7 ligands. In contrast, pertussis toxin-sensitive B cell sticking does not require SLC or MIP-3beta signaling, and occurs efficiently in SLC(low/-) HEV segments in wild-type mice, and in the SLC-negative HEVs of DDD/1 mice. Remarkably, sites of T and B cell firm adhesion are segregated in PPs, with HEVs supporting B cell accumulation concentrated in or near follicles, the target domain of most B cells entering PPs, whereas T cells preferentially accumulate in interfollicular HEVs. Our findings reveal a fundamental difference in signaling requirements for PP-HEV recognition by T and B cells, and describe an unexpected level of specialization of HEVs that may allow differential, segmental control of lymphocyte subset recruitment into functionally distinct lymphoid microenvironments in vivo.

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Desensitization of LNCs with SLC inhibits arrest in PP-HEVs. Unfractionated LNCs (70–85% CD3+ by FACS®) of young mice were washed and incubated in cRPMI for 1 h in tissue culture flasks. LNCs were fluorescently labeled, incubated with chemokine (SLC and SDF-1α) or control medium for 45 min at 37°C, washed through a warm serum gradient to remove residual chemokine, and resuspended together with internal standard cells (labeled in a different color) immediately before injection via carotid artery cannulation into mice prepared for intravital microscopy. Areas within the visualized PPs were surveyed in the initial 2 min for HEVs supporting lymphocyte interactions, and then observed for a further 10 min. Total flux and rolling data (for test and for internal standard cells) were collected during a 2-min period 6–8 min after injection, and represent at least 10 consecutive cells of each type in at least 10 HEVs per preparation. Accumulated cells in these same HEVs were enumerated at the 8-min time point. Lymphocyte total flux, rolling fraction, or accumulation was variable in different vessels and PPs, reflecting the natural variability in hemodynamic parameters and vascular characteristics. Therefore, values determined for experimental cells (chemokine-desensitized or mock-treated cells of one color) were normalized by dividing by values obtained for coinjected internal standard population (labeled in a separate color) determined in the same recipient vessels. The ratio of chemokine-treated cell to internal standard cell values (e.g., rolling fractions), determined in one set of recipients, was then divided by the ratio of mock-treated control cell to internal standard cell values determined in littermate recipients, and multiplied by 100. Thus, the data presented represent the total flux, rolling fraction, or accumulation of chemokine-treated cells expressed as a percentage of those of mock-treated control cells labeled identically and analyzed in parallel. Therefore, 100% in the graph represents the behavior of the mock-treated control cells: for these cells, the total flux of cells entering HEVs in the blood per minute ranged from 16 to 60, mean 36 ± 14 SD in a representative experiment; the rolling fraction ranged from 27 to 100%, mean 69 ± 20% SD; and the number of cells accumulated in HEV segments ranged from 0 to 60 or more by 10 min. These values illustrate the natural variability associated with different vascular segments and observation periods that necessitated the use of internal standard cells for these studies. Rolling velocity ranged from 25 to 170 μm/s, mean 81 ± 46 SD. Mean results of five experimental animals for each treatment (± SD) are shown in the graph. Inhibition of accumulation by SLC treatment is significant compared with SDF-1α or control treatment by Student's t test (P < 0.01).
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Figure 1: Desensitization of LNCs with SLC inhibits arrest in PP-HEVs. Unfractionated LNCs (70–85% CD3+ by FACS®) of young mice were washed and incubated in cRPMI for 1 h in tissue culture flasks. LNCs were fluorescently labeled, incubated with chemokine (SLC and SDF-1α) or control medium for 45 min at 37°C, washed through a warm serum gradient to remove residual chemokine, and resuspended together with internal standard cells (labeled in a different color) immediately before injection via carotid artery cannulation into mice prepared for intravital microscopy. Areas within the visualized PPs were surveyed in the initial 2 min for HEVs supporting lymphocyte interactions, and then observed for a further 10 min. Total flux and rolling data (for test and for internal standard cells) were collected during a 2-min period 6–8 min after injection, and represent at least 10 consecutive cells of each type in at least 10 HEVs per preparation. Accumulated cells in these same HEVs were enumerated at the 8-min time point. Lymphocyte total flux, rolling fraction, or accumulation was variable in different vessels and PPs, reflecting the natural variability in hemodynamic parameters and vascular characteristics. Therefore, values determined for experimental cells (chemokine-desensitized or mock-treated cells of one color) were normalized by dividing by values obtained for coinjected internal standard population (labeled in a separate color) determined in the same recipient vessels. The ratio of chemokine-treated cell to internal standard cell values (e.g., rolling fractions), determined in one set of recipients, was then divided by the ratio of mock-treated control cell to internal standard cell values determined in littermate recipients, and multiplied by 100. Thus, the data presented represent the total flux, rolling fraction, or accumulation of chemokine-treated cells expressed as a percentage of those of mock-treated control cells labeled identically and analyzed in parallel. Therefore, 100% in the graph represents the behavior of the mock-treated control cells: for these cells, the total flux of cells entering HEVs in the blood per minute ranged from 16 to 60, mean 36 ± 14 SD in a representative experiment; the rolling fraction ranged from 27 to 100%, mean 69 ± 20% SD; and the number of cells accumulated in HEV segments ranged from 0 to 60 or more by 10 min. These values illustrate the natural variability associated with different vascular segments and observation periods that necessitated the use of internal standard cells for these studies. Rolling velocity ranged from 25 to 170 μm/s, mean 81 ± 46 SD. Mean results of five experimental animals for each treatment (± SD) are shown in the graph. Inhibition of accumulation by SLC treatment is significant compared with SDF-1α or control treatment by Student's t test (P < 0.01).

Mentions: Desensitization with CCR7 ligand SLC, but not with the CXCR4 ligand SDF-1α, substantially inhibits accumulation of intravenously injected LNCs in PP-HEVs in vivo ( Fig. 1). Total flux and rolling fraction of SLC-treated lymphocytes through HEV were, if anything, slightly increased over control. Rolling velocity ( Fig. 1 legend) and surface levels of adhesion receptors L-selectin, LFA-1, and α4β7 on desensitized lymphocytes (data not shown) remained unchanged from mock-treated control. Chemokine desensitization dramatically reduced the conversion of rolling to sticking behavior and firm arrest, an effect that is similar to PTX treatment and consistent with inhibition of the specific Gαi protein–linked signaling event required for integrin-mediated activated rapid adhesion.


The role of chemokines in the microenvironmental control of T versus B cell arrest in Peyer's patch high endothelial venules.

Warnock RA, Campbell JJ, Dorf ME, Matsuzawa A, McEvoy LM, Butcher EC - J. Exp. Med. (2000)

Desensitization of LNCs with SLC inhibits arrest in PP-HEVs. Unfractionated LNCs (70–85% CD3+ by FACS®) of young mice were washed and incubated in cRPMI for 1 h in tissue culture flasks. LNCs were fluorescently labeled, incubated with chemokine (SLC and SDF-1α) or control medium for 45 min at 37°C, washed through a warm serum gradient to remove residual chemokine, and resuspended together with internal standard cells (labeled in a different color) immediately before injection via carotid artery cannulation into mice prepared for intravital microscopy. Areas within the visualized PPs were surveyed in the initial 2 min for HEVs supporting lymphocyte interactions, and then observed for a further 10 min. Total flux and rolling data (for test and for internal standard cells) were collected during a 2-min period 6–8 min after injection, and represent at least 10 consecutive cells of each type in at least 10 HEVs per preparation. Accumulated cells in these same HEVs were enumerated at the 8-min time point. Lymphocyte total flux, rolling fraction, or accumulation was variable in different vessels and PPs, reflecting the natural variability in hemodynamic parameters and vascular characteristics. Therefore, values determined for experimental cells (chemokine-desensitized or mock-treated cells of one color) were normalized by dividing by values obtained for coinjected internal standard population (labeled in a separate color) determined in the same recipient vessels. The ratio of chemokine-treated cell to internal standard cell values (e.g., rolling fractions), determined in one set of recipients, was then divided by the ratio of mock-treated control cell to internal standard cell values determined in littermate recipients, and multiplied by 100. Thus, the data presented represent the total flux, rolling fraction, or accumulation of chemokine-treated cells expressed as a percentage of those of mock-treated control cells labeled identically and analyzed in parallel. Therefore, 100% in the graph represents the behavior of the mock-treated control cells: for these cells, the total flux of cells entering HEVs in the blood per minute ranged from 16 to 60, mean 36 ± 14 SD in a representative experiment; the rolling fraction ranged from 27 to 100%, mean 69 ± 20% SD; and the number of cells accumulated in HEV segments ranged from 0 to 60 or more by 10 min. These values illustrate the natural variability associated with different vascular segments and observation periods that necessitated the use of internal standard cells for these studies. Rolling velocity ranged from 25 to 170 μm/s, mean 81 ± 46 SD. Mean results of five experimental animals for each treatment (± SD) are shown in the graph. Inhibition of accumulation by SLC treatment is significant compared with SDF-1α or control treatment by Student's t test (P < 0.01).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195795&req=5

Figure 1: Desensitization of LNCs with SLC inhibits arrest in PP-HEVs. Unfractionated LNCs (70–85% CD3+ by FACS®) of young mice were washed and incubated in cRPMI for 1 h in tissue culture flasks. LNCs were fluorescently labeled, incubated with chemokine (SLC and SDF-1α) or control medium for 45 min at 37°C, washed through a warm serum gradient to remove residual chemokine, and resuspended together with internal standard cells (labeled in a different color) immediately before injection via carotid artery cannulation into mice prepared for intravital microscopy. Areas within the visualized PPs were surveyed in the initial 2 min for HEVs supporting lymphocyte interactions, and then observed for a further 10 min. Total flux and rolling data (for test and for internal standard cells) were collected during a 2-min period 6–8 min after injection, and represent at least 10 consecutive cells of each type in at least 10 HEVs per preparation. Accumulated cells in these same HEVs were enumerated at the 8-min time point. Lymphocyte total flux, rolling fraction, or accumulation was variable in different vessels and PPs, reflecting the natural variability in hemodynamic parameters and vascular characteristics. Therefore, values determined for experimental cells (chemokine-desensitized or mock-treated cells of one color) were normalized by dividing by values obtained for coinjected internal standard population (labeled in a separate color) determined in the same recipient vessels. The ratio of chemokine-treated cell to internal standard cell values (e.g., rolling fractions), determined in one set of recipients, was then divided by the ratio of mock-treated control cell to internal standard cell values determined in littermate recipients, and multiplied by 100. Thus, the data presented represent the total flux, rolling fraction, or accumulation of chemokine-treated cells expressed as a percentage of those of mock-treated control cells labeled identically and analyzed in parallel. Therefore, 100% in the graph represents the behavior of the mock-treated control cells: for these cells, the total flux of cells entering HEVs in the blood per minute ranged from 16 to 60, mean 36 ± 14 SD in a representative experiment; the rolling fraction ranged from 27 to 100%, mean 69 ± 20% SD; and the number of cells accumulated in HEV segments ranged from 0 to 60 or more by 10 min. These values illustrate the natural variability associated with different vascular segments and observation periods that necessitated the use of internal standard cells for these studies. Rolling velocity ranged from 25 to 170 μm/s, mean 81 ± 46 SD. Mean results of five experimental animals for each treatment (± SD) are shown in the graph. Inhibition of accumulation by SLC treatment is significant compared with SDF-1α or control treatment by Student's t test (P < 0.01).
Mentions: Desensitization with CCR7 ligand SLC, but not with the CXCR4 ligand SDF-1α, substantially inhibits accumulation of intravenously injected LNCs in PP-HEVs in vivo ( Fig. 1). Total flux and rolling fraction of SLC-treated lymphocytes through HEV were, if anything, slightly increased over control. Rolling velocity ( Fig. 1 legend) and surface levels of adhesion receptors L-selectin, LFA-1, and α4β7 on desensitized lymphocytes (data not shown) remained unchanged from mock-treated control. Chemokine desensitization dramatically reduced the conversion of rolling to sticking behavior and firm arrest, an effect that is similar to PTX treatment and consistent with inhibition of the specific Gαi protein–linked signaling event required for integrin-mediated activated rapid adhesion.

Bottom Line: We show that T cells roll on most Peyer's patch high endothelial venules (PP-HEVs), but preferentially arrest in segments displaying high levels of luminal secondary lymphoid tissue chemokine (SLC) (6Ckine, Exodus-2, thymus-derived chemotactic agent 4 [TCA-4]).Remarkably, sites of T and B cell firm adhesion are segregated in PPs, with HEVs supporting B cell accumulation concentrated in or near follicles, the target domain of most B cells entering PPs, whereas T cells preferentially accumulate in interfollicular HEVs.Our findings reveal a fundamental difference in signaling requirements for PP-HEV recognition by T and B cells, and describe an unexpected level of specialization of HEVs that may allow differential, segmental control of lymphocyte subset recruitment into functionally distinct lymphoid microenvironments in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, Department of Pathology. Stanford University Medical School, Stanford, California, USA.

ABSTRACT
Chemokines have been hypothesized to contribute to the selectivity of lymphocyte trafficking not only as chemoattractants, but also by triggering integrin-dependent sticking (arrest) of circulating lymphocytes at venular sites of extravasation. We show that T cells roll on most Peyer's patch high endothelial venules (PP-HEVs), but preferentially arrest in segments displaying high levels of luminal secondary lymphoid tissue chemokine (SLC) (6Ckine, Exodus-2, thymus-derived chemotactic agent 4 [TCA-4]). This arrest is selectively inhibited by functional deletion (desensitization) of CC chemokine receptor 7 (CCR7), the receptor for SLC and for macrophage inflammatory protein (MIP)-3beta (EBV-induced molecule 1 ligand chemokine [ELC]), and does not occur in mutant DDD/1 mice that are deficient in these CCR7 ligands. In contrast, pertussis toxin-sensitive B cell sticking does not require SLC or MIP-3beta signaling, and occurs efficiently in SLC(low/-) HEV segments in wild-type mice, and in the SLC-negative HEVs of DDD/1 mice. Remarkably, sites of T and B cell firm adhesion are segregated in PPs, with HEVs supporting B cell accumulation concentrated in or near follicles, the target domain of most B cells entering PPs, whereas T cells preferentially accumulate in interfollicular HEVs. Our findings reveal a fundamental difference in signaling requirements for PP-HEV recognition by T and B cells, and describe an unexpected level of specialization of HEVs that may allow differential, segmental control of lymphocyte subset recruitment into functionally distinct lymphoid microenvironments in vivo.

Show MeSH
Related in: MedlinePlus