The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules - PubMed (original) (raw)

The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules

J V Stein et al. J Exp Med. 2000.

Abstract

T cell homing to peripheral lymph nodes (PLNs) is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial tethering and rolling via L-selectin, firm adhesion of T cells requires rapid upregulation of lymphocyte function-associated antigen 1 (LFA-1) adhesiveness by a previously unknown pathway that activates a Galpha(i)-linked receptor. Here, we used intravital microscopy of murine PLNs to study the role of thymus-derived chemotactic agent (TCA)-4 (secondary lymphoid tissue chemokine, 6Ckine, Exodus-2) in homing of adoptively transferred T cells from T-GFP mice, a transgenic strain that expresses green fluorescent protein (GFP) selectively in naive T lymphocytes (T(GFP) cells). TCA-4 was constitutively presented on the luminal surface of HEVs, where it was required for LFA-1 activation on rolling T(GFP) cells. Desensitization of the TCA-4 receptor, CC chemokine receptor 7 (CCR7), blocked T(GFP) cell adherence in wild-type HEVs, whereas desensitization to stromal cell-derived factor (SDF)-1alpha (the ligand for CXC chemokine receptor 4 [CXCR4]) did not affect T(GFP) cell behavior. TCA-4 protein was not detected on the luminal surface of PLN HEVs in plt/plt mice, which have a congenital defect in T cell homing to PLNs. Accordingly, T(GFP) cells rolled but did not arrest in plt/plt HEVs. When TCA-4 was injected intracutaneously into plt/plt mice, the chemokine entered afferent lymph vessels and accumulated in draining PLNs. 2 h after intracutaneous injection, luminal presentation of TCA-4 was detectable in a subset of HEVs, and LFA-1-mediated T(GFP) cell adhesion was restored in these vessels. We conclude that TCA-4 is both required and sufficient for LFA-1 activation on rolling T cells in PLN HEVs. This study also highlights a hitherto undocumented role for chemokines contained in afferent lymph, which may modulate leukocyte recruitment in draining PLNs.

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Figures

Figure 1

Figure 1

Expression of PNAd and TCA-4 in wild-type and plt/plt PLNs. Serial sections (9 μm) of wild-type (A and B) or plt/plt PLNs (C and D) were stained for PNAd (mAb MECA-79; A and C) or TCA-4 (mAbs 3D5 and 4B1; B and D). PNAd (A) and TCA-4 (B) are colocalized in wild-type HEVs. Although PNAd expression is normal in plt/plt HEVs (C), no TCA-4 can be detected in these animals (D). Original magnification: ×1,000.

Figure 2

Figure 2

TCA-4 is presented on the luminal surface of wild-type, but not plt/plt, venules in PLNs. Fluorescent microvessels were recorded in anesthetized wild-type and plt/plt mice after intravenous injection of 75 μg anti–mouse TCA-4 mAb FITC-4B1. (A) PLN microvessels in a wild-type mouse. Note that fluorescent anti–TCA-4 delineates the HEV, but not an adjacent arteriole (ART). (B) Skin venules (VEN) or arterioles (ART) in the same wild-type mouse did not stain with anti–TCA-4. (C) FITC-4B1 did not accumulate in PLN venules of plt/plt mice. The extravascular bright spots were already present before mAb injection. These spots are most likely autofluorescent cells in the superficial cortex that are occasionally encountered in wild-type as well as plt/plt PLNs. Similar results were obtained in two other wild-type and plt/plt preparations. Bar, 100 μm. Digitized QuickTime™ videos showing characteristic scenes from intravital TCA-4 staining in wild-type and plt/plt mice (corresponding to A and C, respectively) are available at http://www.jem.org/cgi/content/full/191/1/61/F2/DC1.

Figure 2

Figure 2

TCA-4 is presented on the luminal surface of wild-type, but not plt/plt, venules in PLNs. Fluorescent microvessels were recorded in anesthetized wild-type and plt/plt mice after intravenous injection of 75 μg anti–mouse TCA-4 mAb FITC-4B1. (A) PLN microvessels in a wild-type mouse. Note that fluorescent anti–TCA-4 delineates the HEV, but not an adjacent arteriole (ART). (B) Skin venules (VEN) or arterioles (ART) in the same wild-type mouse did not stain with anti–TCA-4. (C) FITC-4B1 did not accumulate in PLN venules of plt/plt mice. The extravascular bright spots were already present before mAb injection. These spots are most likely autofluorescent cells in the superficial cortex that are occasionally encountered in wild-type as well as plt/plt PLNs. Similar results were obtained in two other wild-type and plt/plt preparations. Bar, 100 μm. Digitized QuickTime™ videos showing characteristic scenes from intravital TCA-4 staining in wild-type and plt/plt mice (corresponding to A and C, respectively) are available at http://www.jem.org/cgi/content/full/191/1/61/F2/DC1.

Figure 3

Figure 3

T lymphocytes from T-GFP transgenic mice specifically express GFP and serve as a useful tool for in vivo studies of T cell homing to PLNs. (A) FACS® analysis of splenocytes and PLN lymphocytes derived from T-GFP transgenic mice. Naive T, but not B cells from T-GFP transgenic mice specifically express GFP. (B) TGFP homing to PLNs and spleen in wild-type and plt/plt mice. The distribution of TGFP cells was analyzed 2 h after tail vein injection and expressed as percentage of GFP+ cells among gated lymphocytes in each lymphoid organ and in systemic blood. One representative experiment (out of six performed with similar results) is shown for each recipient strain.

Figure 3

Figure 3

T lymphocytes from T-GFP transgenic mice specifically express GFP and serve as a useful tool for in vivo studies of T cell homing to PLNs. (A) FACS® analysis of splenocytes and PLN lymphocytes derived from T-GFP transgenic mice. Naive T, but not B cells from T-GFP transgenic mice specifically express GFP. (B) TGFP homing to PLNs and spleen in wild-type and plt/plt mice. The distribution of TGFP cells was analyzed 2 h after tail vein injection and expressed as percentage of GFP+ cells among gated lymphocytes in each lymphoid organ and in systemic blood. One representative experiment (out of six performed with similar results) is shown for each recipient strain.

Figure 4

Figure 4

TGFP cells roll in both wild-type and plt/plt PLNs, but only undergo firm adhesion in wild-type PLN venules. Using intravital microscopy of murine subiliac PLNs, the behavior of TGFP cells was analyzed. (A) Rolling fractions (determined as the percentage of rolling cells in all cells passing through a given venule) of TGFP cells in wild-type (+/+) and plt/plt PLN venules are shown as a function of vessel diameter. Each symbol represents a single venule. (B) Mean rolling fractions of TGFP cells in wild-type and plt/plt HEVs. Rolling fractions are slightly elevated in plt/plt mice (*P < 0.022). (C) Sticking fractions (percentage of rolling cells that arrest for at least 20 s) of TGFP cells in wild-type and plt/plt PLN venules are shown as a function of vessel diameter. (D) Mean sticking fractions of TGFP cells in wild-type and plt/plt PLN venules. Sticking is virtually absent in plt/plt PLN HEVs (*P < 0.0001). Data in B and D are shown as mean ± SEM; n = no. of animals/venules analyzed. Digitized QuickTime™ videos showing characteristic scenes from intravital microscopy experiments of TGFP cell behavior in wild-type and plt/plt mice are available at http://www.jem.org/cgi/content/full/191/1/61/F4/DC1.

Figure 5

Figure 5

Desensitization of CCR7, but not CXCR4, in TGFP cells blocks firm adhesion in wild-type PLN venules. TGFP cells were desensitized by incubation for ≥40 min with 1 μM TCA-4 (agonist for CCR7) or 1 μm SDF-1α (agonist for CXCR4). Desensitized cells were sequentially injected into wild-type mice and observed in identical HEVs. (A) Mean rolling fractions of TCA-4– and SDF-1α–desensitized TGFP cells. There was no statistically significant difference in rolling fractions. (B) Mean sticking fraction of TCA-4– and SDF-1α–desensitized TGFP cells. TGFP cells desensitized to TCA-4, but not SDF-1α, failed to undergo firm adhesion in wild-type PLN venules (*P < 0.03). Data are shown as mean ± SEM; n = no. of animals/venules analyzed.

Figure 6

Figure 6

Intracutaneously injected TCA-4 specifically accumulates in draining PLNs, where it is presented on the luminal surface of HEVs. (A) 125I–TCA-4 (2.6 ng in 50 μl saline) was injected into the skin over the left anterior hind leg of BALB/c mice, and radioactivity was determined at time points specified. The activity of 125I–TCA-4 per mass unit organ was determined in draining (left) versus contralateral (right) subiliac LN and MLN as described in Materials and Methods. Three mice per time point after injection were analyzed. 125I–TCA-4 specifically accumulated in the draining LN, whereas the contralateral LN and MLN remained largely unaffected. (B) Intravital micrograph of fluorescent anti–TCA-4 mAb FITC-4B1 staining (30 min after injection of the mAb) in a branched HEV of a draining subiliac LN of a plt/plt mouse 2 h after intracutaneous injection of TCA-4. 2.5 μg TCA-4 was injected intracutaneously in the left hind leg, and the draining subiliac LN was observed after intraarterial injection of 75 μg anti–TCA-4 mAb FITC-4B1. FITC-4B1 accumulated in some, but not all PLN microvessels. Note that the right venular branch is only partially labeled, and capillaries are not stained at all (see C). An interacting TGFP cell can be seen in the right venular branch (arrow). A digitized QuickTime™ video of this scene is available at http://www.jem.org/cgi/content/full/191/1/61/F6/DC1\. (C) Micrograph of the same field of view as in B after injection of FITC-dextran (150 kD) as a plasma marker. Note that FITC-dextran, unlike anti–TCA-4 mAb, delineates all capillaries (CAP) and venules, including the entire length of the HEV that enters the field of view from the right margin. (D) A skin venule in the same preparation 2 h after intracutaneous injection of TCA-4 and 30 min after injection of anti–TCA-4 mAb FITC-4B1. No specific fluorescence is associated with these nonlymphoid vessels. (E) Micrograph of the same field of view as in D after injection of FITC-dextran. Bar, 50 μm.

Figure 6

Figure 6

Intracutaneously injected TCA-4 specifically accumulates in draining PLNs, where it is presented on the luminal surface of HEVs. (A) 125I–TCA-4 (2.6 ng in 50 μl saline) was injected into the skin over the left anterior hind leg of BALB/c mice, and radioactivity was determined at time points specified. The activity of 125I–TCA-4 per mass unit organ was determined in draining (left) versus contralateral (right) subiliac LN and MLN as described in Materials and Methods. Three mice per time point after injection were analyzed. 125I–TCA-4 specifically accumulated in the draining LN, whereas the contralateral LN and MLN remained largely unaffected. (B) Intravital micrograph of fluorescent anti–TCA-4 mAb FITC-4B1 staining (30 min after injection of the mAb) in a branched HEV of a draining subiliac LN of a plt/plt mouse 2 h after intracutaneous injection of TCA-4. 2.5 μg TCA-4 was injected intracutaneously in the left hind leg, and the draining subiliac LN was observed after intraarterial injection of 75 μg anti–TCA-4 mAb FITC-4B1. FITC-4B1 accumulated in some, but not all PLN microvessels. Note that the right venular branch is only partially labeled, and capillaries are not stained at all (see C). An interacting TGFP cell can be seen in the right venular branch (arrow). A digitized QuickTime™ video of this scene is available at http://www.jem.org/cgi/content/full/191/1/61/F6/DC1\. (C) Micrograph of the same field of view as in B after injection of FITC-dextran (150 kD) as a plasma marker. Note that FITC-dextran, unlike anti–TCA-4 mAb, delineates all capillaries (CAP) and venules, including the entire length of the HEV that enters the field of view from the right margin. (D) A skin venule in the same preparation 2 h after intracutaneous injection of TCA-4 and 30 min after injection of anti–TCA-4 mAb FITC-4B1. No specific fluorescence is associated with these nonlymphoid vessels. (E) Micrograph of the same field of view as in D after injection of FITC-dextran. Bar, 50 μm.

Figure 7

Figure 7

Intracutaneously injected TCA-4, but not SDF-1α, reconstitutes TGFP cell sticking in plt/plt PLN venules. 1 μg TCA-4 or SDF-1α was injected intracutaneously in left hind legs of plt/plt mice. Surgical preparation of the draining left inguinal LN was started 30–40 min after chemokine injection. TGFP cell behavior was observed in the draining subiliac PLN 90 min after chemokine injection using intravital microscopy. (A) Rolling fractions of TGFP cells in plt/plt PLN venules without chemokine injection (+) or after injection of 1 μg TCA-4 (▴) or SDF-1α (⋄) as a function of vessel diameter. (B) Mean rolling fractions of TGFP cells in plt/plt PLN venules. The rolling frequency was not affected by chemokines. (C–E) Sticking fractions of TGFP cells in plt/plt PLN venules without chemokine injection (C) or after pretreatment with TCA-4 (D) or SDF-1α (E) as a function of the vessel diameter. (F) Mean sticking fractions of TGFP cells in plt/plt PLN venules. TGFP cells underwent firm adhesion in some, but not all, HEVs of TCA-4–pretreated plt/plt mice (*P < 0.008 vs. no chemokine, and P < 0.02 vs. SDF-1α–treated animals). On the other hand, SDF-1α–pretreated plt/plt PLN venules did not support firm adhesion of TGFP cells, except in one animal (out of five) where some sticking was seen in two venules (P = 0.34 vs. no chemokine). Data are shown as mean ± SEM; n = no. of animals/venules analyzed. A digitized QuickTime™ video showing T cell rolling and sticking in an HEV of a plt/plt mouse 1 h after intracutaneous injection of TCA-4 is available at http://www.jem.org/cgi/content/full/191/1/61/F7/DC1.

Figure 8

Figure 8

TCA-4–induced firm arrest of TGFP cells in plt/plt HEVs is LFA-1 dependent. Mean sticking fractions of TGFP cells in plt/plt HEVs pretreated with TCA-4 before and after anti–LFA-1 mAb (TIB 213) treatment. After assessment of TGFP cell sticking in the absence of mAb (control), mice and GFP-T cells were treated with anti–LFA-1 mAb and observed in the same HEVs that supported firm arrest of control cells. In contrast to untreated TGFP cells, anti–LFA-1 mAb–treated TGFP cells failed to become firmly adherent (*P < 0.05). Data are shown as mean ± SEM; n = no. of animals/venules analyzed.

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