Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs

Abstract

The extracellular lysophospholipase D autotaxin (ATX) and its product, lysophosphatidic acid, have diverse functions in development and cancer, but little is known about their functions in the immune system. Here we found that ATX had high expression in the high endothelial venules of lymphoid organs and was secreted. Chemokine-activated lymphocytes expressed receptors with enhanced affinity for ATX, which provides a mechanism for targeting the secreted ATX to lymphocytes undergoing recruitment. Lysophosphatidic acid induced chemokinesis in T cells. Intravenous injection of enzymatically inactive ATX attenuated the homing of T cells to lymphoid tissues, probably through competition with endogenous ATX and exertion of a dominant negative effect. Our results support the idea of a new and general step in the homing cascade in which the ectoenzyme ATX facilitates the entry of lymphocytes into lymphoid organs.

Figure 1

Expression of ATX transcripts in mouse tissues. (a) First strand cDNA was synthesized from RNA obtained from mouse tissues and subjected to quantitative PCR analysis. The abundance of ATX mRNA in each tissue was normalized to HPRT mRNA, and related to the normalized level in mesenteric lymph node (MLN). The tissues (or cell lines) analyzed were: PLN, peripheral lymph node; PP, Peyer’s patch; HECs, high endothelial cells; SPL, spleen; THY, thymus; SC, spinal cord; MG, mammary gland; SI, small intestine; BM, bone marrow; bEnd.3, mouse endothelial cell line; SVEC, mouse endothelial cell line. The data represent means and SD’s from measurements performed on 3 independent animals. (b) ATX mRNA expression in PLN and spleen was determined by in situ hybridization using an ³⁵S-labeled mouse ATX antisense probe. Specific signals were not present in the sense controls (not shown). These results are representative of two separate experiments. In both experiments at least one lymphoid organ of each type was examined at three exposure times. Bar denotes 100 µm. RP, red pulp; WP, white pulp.

Figure 2

Localization of ATX protein in lymphoid organs. Two-color immunofluorescence was performed on cryostat sections of PLN and MLN for ATX (green) and MECA-79 reactivity (red) and on spleen sections for ATX (green) and CD31 (red). Bright field images of the same fields (hematoxylin staining) are presented on the right. Scale bars, 100 µm. White arrows indicate ATX-positive small vessels, which are negative for MECA-79. The results shown are representative of 4 independent experiments.

Figure 3

Secretion of ATX and HECs and transfected MDCK cells. (a) Conditioned medium from isolated HECs was subjected to SDS-PAGE and immunoblotted for ATX and GlyCAM-1 The membrane was stripped and reprobed with normal rabbit IgG as a control. The results shown are representative of three independent experiments. (b) MDCK monolayers (ATX-transfected or mock-transfected) were evaluated for integrity by measuring diffusion of FITC-dextran across the monolayers in transwell units. Diffusion across a bare filter is shown for comparison. (c,d) Supernatants were collected from above (apical) or below (basal) the MDCK monolayers. In c, the supernatants were immunoblotted for ATX or clusterin (Clstr) or with control mouse IgG. In d, the supernatants were analyzed for ATX enzymatic activity (lysoPLD). The slight difference in molecular weight between rATX and native ATX in a is likely due to glycosylation differences. The results in b, c and d are representative of two independent experiments.

Figure 4

Integrin dependency of ATX binding to human T cells. (a) The interaction of ATX with Jurkat T cells was examined by a static adhesion assay using ATX-coated plastic wells (at the indicated input concentration) in divalent cation-free Hanks’ balanced salt solution with or without 0.5 mM Mn²⁺. sICAM and sVCAM (human, 200 ng) were coated onto wells to serve as positive controls for adhesive substrates. Results are shown as means ± SD’s. of triplicate determinations. (b) Jurkat T cells were pretreated with 5 µg/ml of function-blocking antibodies for integrin α_M, α₄, β₁ or β₂ in Mn²⁺ containing buffer and then tested for adhesion to ATX (200 ng) in the presence of Mn²⁺. Two separate experiments are presented in the left and right panels. Adhesion to VCAM-1-coated wells (200 ng) is shown for comparison. Results are presented as means ± SD’s. of triplicate determinations. (c) The interaction of ATX with CCL21-stimulated Jurkat T cells was examined by static adhesion assay. The cells were allowed to interact with CCL21 (100 ng) co-immobilized with ATX protein (200 ng) on the plate. Results are shown as means ± SD’s of triplicate determinations. (d) Human T cell binding was measured to ATX- or VCAM-1-coated wells at the indicated substrate concentration in the presence of Mn²⁺. Results are shown as means ± SD’s. of triplicate determinations. (e) Function-blocking antibodies against integrin subunits were tested for their effects on the adhesion of human T-cells to wells coated with ATX (200 ng) in the presence of Mn²⁺. Adhesion to VCAM-1 (200 ng) is shown for comparison. Results are presented as means ± SD’s. of triplicate determinations. (f) ATX interaction with primary human T cells. Human T cells were exposed to co-immobilized CCL21 and ATX as above. Adhesion was tested after treating the cells with function-blocking antibodies as indicated. Results are shown as means ± SD’s of triplicate determinations. * denotes that the inhibitory effect of the antibody relative to the control was significant with a P value of < 0.01. The results in a–c are representative of 3–4 and those of d and f 2 experiments. The experiment presented in e was performed once.

Figure 4

Integrin dependency of ATX binding to human T cells. (a) The interaction of ATX with Jurkat T cells was examined by a static adhesion assay using ATX-coated plastic wells (at the indicated input concentration) in divalent cation-free Hanks’ balanced salt solution with or without 0.5 mM Mn²⁺. sICAM and sVCAM (human, 200 ng) were coated onto wells to serve as positive controls for adhesive substrates. Results are shown as means ± SD’s. of triplicate determinations. (b) Jurkat T cells were pretreated with 5 µg/ml of function-blocking antibodies for integrin α_M, α₄, β₁ or β₂ in Mn²⁺ containing buffer and then tested for adhesion to ATX (200 ng) in the presence of Mn²⁺. Two separate experiments are presented in the left and right panels. Adhesion to VCAM-1-coated wells (200 ng) is shown for comparison. Results are presented as means ± SD’s. of triplicate determinations. (c) The interaction of ATX with CCL21-stimulated Jurkat T cells was examined by static adhesion assay. The cells were allowed to interact with CCL21 (100 ng) co-immobilized with ATX protein (200 ng) on the plate. Results are shown as means ± SD’s of triplicate determinations. (d) Human T cell binding was measured to ATX- or VCAM-1-coated wells at the indicated substrate concentration in the presence of Mn²⁺. Results are shown as means ± SD’s. of triplicate determinations. (e) Function-blocking antibodies against integrin subunits were tested for their effects on the adhesion of human T-cells to wells coated with ATX (200 ng) in the presence of Mn²⁺. Adhesion to VCAM-1 (200 ng) is shown for comparison. Results are presented as means ± SD’s. of triplicate determinations. (f) ATX interaction with primary human T cells. Human T cells were exposed to co-immobilized CCL21 and ATX as above. Adhesion was tested after treating the cells with function-blocking antibodies as indicated. Results are shown as means ± SD’s of triplicate determinations. * denotes that the inhibitory effect of the antibody relative to the control was significant with a P value of < 0.01. The results in a–c are representative of 3–4 and those of d and f 2 experiments. The experiment presented in e was performed once.

Figure 5

LPA effects on human T cells. (a) T cells were stimulated by the indicated amount of LPA or CXCL12 at 250 ng/ml for 37 °C for 15 min, fixed and permeabilized and then stained with Alexa 488-conjugated phalloidin. The amount of F-actin is shown normalized to that in untreated cells. (b) T cells were added to the upper chamber of transwell units with LPA added either to the same chamber (to induce chemokinesis) or to the lower chamber (to induce chemotaxis). After 3 h of incubation at 37 °C, cells that had migrated to the bottom chamber were quantified by flow cytometry. CXCL12 (250 ng/ml in the lower chamber) was used as a positive control for a lymphocyte chemoattractant. (c) T cells were either pretreated with PTX or not and then tested for their chemokinetic response to LPA in same assay as above. (d) LPA was added at 1 µM to the upper, lower, or to both chambers and tested for its effects on T cell migration from the upper to lower chamber after 3 h. (e) The transwell assay was performed as above with LPA in the upper chamber (1 µM) and/or CXCL12 (50 ng/ml) in the lower chamber. In all experiments, results are presented as means ± SD’s of triplicate determinations. (f) Human T cell migration assay was performed with 1 µM of LPA in the upper chamber (1 µM) and/or CCL21 (50 ng/ml) in the lower chamber. In all experiments (a–f), results are presented as means ± SD’s of triplicate determinations. The * indicates that the combination of chemokine (CXCL12 or CCL21) and LPA induced more T cell migration than chemokine alone (P < 0.01). (g) Chemokine receptor expression of LPA-stimulated T cells was tested by flow cytometry. T cells were incubated with indicated concentration of LPA at 37 °C for 3 h. The cell surface receptors were detected with anti-CXCR4 or anti-CCR7 mAb. The results in a, b, and g are representative of two independent experiments. The experiments in c, d, e, and f were each performed once.

Figure 6

Migration of T cells to and within lymphoid organs in the presence of exogenous ATX. (a) CFSE-labeled T cells were pretreated with PBS, active ATX (WT) or inactive ATX (T210A) and the mixtures were injected intravenously into recipient mice. The amount of ATX was 4 µg per animal. 15 min later, mice were sacrificed and fluorescent cells in each tissue were quantified by flow cytometry. The relative homing indexes are shown as means ± SD’s with results pooled from two experiments. For the lymphoid organs, n = 6 for PBS, and n = 8 for both WT and inactive ATX. For blood, n = 4 for each condition. * signify P values of <0.01 as compared to PBS controls. The results are representative of 5 experiments. (b) The entry of lymphocytes into PLNs was independently determined by counting the number of fluorescent lymphocytes present in cryostat sections. As above, lymph nodes were processed 15 min after injection of labeled lymphocytes. Total lymphocytes were counted in at least 10 non-sequential sections for each treatment with three animals per treatment (PBS, WT ATX or T210 ATX). The results are shown as means ± SD’s and are representative of two independent experiments. (c) Lymphocyte migration within PLNs was determined by measuring the distance between extravasated lymphocytes (outside of HEVs) and the nearest HEV. With the same treatments as above, lymph nodes were processed 15 min after injection of fluorescent lymphocytes. HEVs were identified by staining with MECA-79. The upper three panels show representative sections for the three treatments. The bottom graph shows all of the individual measurements taken from 10 non-sequential sections from 3 independent animals per treatment. The data are representative of two independent experiments. * denotes a P value of <0.05