Effector T cells are the principal mediators of acute allograft rejection. These cells may arise from naïve or central memory T cells activated within secondary lymphoid organs (e.g., lymph nodes) or they may develop from effector memory T cells directly recruited from blood and activated within peripheral tissues such as an allograft (1, 2). Recruitment of effector memory (or effector) T cells is initiated by alterations in the graft endothelial cells (ECs), specifically expression of cytokine-inducible adhesion molecules and display of proinflammatory chemokines (reviewed in ). Proinflammatory chemokines, which bind to CXCR3 expressed on memory T cells, notably interferon (IFN)-γ-inducible protein of 10 kDa (IP-10; CXCL10), have been shown to be particularly important for recruitment of T cells that mediate rejection (4–7).
Several discrete steps in T-cell recruitment have been observed using intravital microscopy and dissected in vitro using cytokine-treated EC monolayers. This multistep process involves: (a) tethering and rolling, mediated by rapidly formed and broken interactions between stationary EC and T cells propelled by flowing blood; (b) activation of T-cell integrins mediated by chemokines displayed on the EC surface; (c) firm adhesion of T cells to the ECs; and (d) transendothelial migration (TEM) (3). Molecules that mediate the low affinity interactions involved in T-cell tethering and rolling include selectins and their ligands (reviewed in (8)) and/or T-cell very late antigen (VLA)-4 integrin (CD49d/CD29) in its unactivated state binding to endothelial vascular cell adhesion molecule (VCAM)-1 (CD106) (9). Firm adhesion is mediated by chemokine-activated lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) or VLA-4 integrin binding to endothelial intercellular adhesion molecule (ICAM)-1 (CD54) or VCAM-1, respectively (reviewed in ). Rapid TEM of T cells requires display of chemokine on the endothelial surface and, like rolling, requires shear force imparted by flowing blood (11). A surprising discrepancy between in vivo observations and in vitro modeling is that IP-10 has appeared ineffective at stimulating TEM of freshly isolated CXCR3+ human peripheral blood T cells in vitro (12–14) yet the same cells readily respond to other chemokines, notably stromal cell-derived factor-1α (SDF-1α), a mediator of homeostatic migration (11, 15). IP-10 will stimulate TEM of peripheral blood T cells cultured for 5-7 days in interleukin (IL)-2 (13, 14), a procedure that activates these cells in an undefined manner. Here we report a simple resolution of this seeming paradox, namely that effector memory T cells do undergo rapid TEM in response to IP-10 but that detection of their behavior in vitro requires prior enrichment for this type of T cell, which constitutes only a small fraction of the total T cell population. We also show that cytokine treatment of the EC can be effectively replaced by transduction with a single adhesion molecule, either VCAM-1 or ICAM-1.
MATERIALS AND METHODS
Human umbilical vein ECs (HUVECs) were prepared and cultured as described (16). All HUVECs were used at no later than subculture five; all cells in a given experiment were used at the same level of subculture. Where indicated, HUVECs were incubated in the presence of 10 ng/ml recombinant human tumor necrosis factor (TNF) for 20-26 hr. Alternatively, HUVECs were transduced with retroviral supernatants prepared from Phoenix cells transfected with pLZRS retroviral vectors containing either the human E- selectin, ICAM-1, or VCAM-1 cDNA in the multiple cloning site, as described previously (17). For the E-selectin and ICAM-1 constructs, a cytomegalovirus (CMV) early promoter/enhancer was placed directly upstream of the cDNA.
CD4+ T cells were isolated by positive selection with magnetic beads (Dynal) from PBMCs prepared by Ficoll gradient of blood collected from healthy donors. Memory CD4+ T cells were isolated by depletion of CD45RA+ cells from CD4+ T cells. Central memory and effector memory CD4+ T subsets were prepared from memory CD4+ T cells by using CD62L (Pharmingen) and CCR7 (R&D Systems) monoclonal antibodies and the CELLection pan-mouse IgG kit (Dynal) by positive selection, and negative selection, respectively. “Freshly isolated” CD4+ T cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, penicillin/streptomycin, and nonessential amino acids, overnight, prior to assays. IL-2 “activated” CD4+ T cells were cultured in the same medium containing 400 U/ml recombinant human IL-2 (from the BRB Preclinical Repository, NCI) and 50 μM β-mercaptoethanol for 5-7 days.
The expression of E-selectin, ICAM-1, and VCAM-1 in TNF-treated or transduced HUVECs were determined by FACS analysis (FACSort, Becton Dickinson) after immunostaining with monoclonal antibodies H4/18 (E-Selectin) or E16.5 (VCAM-1) followed by FITC-conjugated goat anti-mouse, or by CD54-FITC (ICAM-1; Immunotech), or with isotype-matched control antibodies. FITC or PE-conjugated antibodies to CD45R0, CD45RA, CD62L (from Immunotech), and CXCR3 (from Pharmingen) were used for CD4+ T cell characterization.
Transendothelial Migration Assays under Conditions of Physiological Shear Stress
HUVECs, grown to confluence on 35-mm fibronectin-coated coverglasses, were washed twice with RPMI/10% FBS, overlaid or not with chemokine (R&D Systems) in RPMI/10% FBS for 5 min, washed two more times, and assembled with a parallel plate flow chamber apparatus (Glycotech) using the 0.01-inch high, 5-mm wide slit gasket provided by the manufacturer. CD4+ T cells were prelabeled with 0.3 μg/ml calcein (Molecular Probes) for one hour in PBS containing 1 mM calcium chloride and 0.5 mM magnesium chloride, washed, and resuspended in culture medium at approximately 2 million cells/ml. On a 37°C heating surface, CD4+ T cells (500 μl) suspended in the same medium were loaded onto the HUVEC monolayer at 0.75 dyne/cm2 for 2 min, followed by medium only at 0 or 1 dyne/cm2 for 15 min. Samples were then fixed with 4% paraformaldehyde, mounted on slides using mounting medium containing DAPI, and examined by microscopy. A FITC filter was used to detect calcein-labeled CD4+ T cells and a DAPI filter used to detect nuclei. Phase contrast optics were used to determine whether CD4+ T cells were either on top or underneath the HUVEC monolayer. CD4+ T cells that were captured and not spread were round and bright when viewed under phase contrast. CD4+ T cells that were spread, but still on top of the HUVEC monolayer were surrounded by a corona of light, while those that had transmigrated had at least one dark edge. The total number of captured CD4+ T cells (i.e., T cells adhered to the apical EC surface and the transmigrated T cells) and the number of transmigrated CD4+ T cells were counted in each 40× field; ten fields were analyzed for each sample.
Statistical significance was determined by one-way analysis of variance using a 95% confidence interval and the Tukey post test (Prism 4.0 for Macintosh). Statistical error is expressed as SEM.
In agreement with a previous report (11), freshly isolated human peripheral blood CD4+ T cells adhere to TNF-treated HUVEC monolayers but do not rapidly transmigrate across the EC monolayer even when subjected to a shear force of 1 dyne/cm2 unless the ECs are first overlaid with the chemokine SDF-1α. The chemokine IP-10 appears ineffective in this assay unless the T cells are first cultured in IL-2 for 5-7 days (Fig. 1). We then fractionated the CD4+ T cell population to isolate CD45RO+ memory cells or further separated these cells into CD62Lhi CCR7hi central memory cells and CD62Llo CCR7lo effector memory cells (Fig. 2A) and repeated this experiment. When we analyzed these subsets for TEM, we observed that the total memory cell subset is slightly enriched for IP-10-responsive cells, and all of the transmigrating memory cells are found within the effector memory subset (Fig. 2B). The effector memory subset constituted less than 10% of the total CD4+ T cell population and these cells were too few for their behavior to be apparent prior to enrichment. Although effector memory cells are enriched for expression of CXCR3, the receptor for IP-10, at least 50% of the unresponsive central memory cells express this receptor at the same levels as found on effector memory cells (Fig. 2A). TEM of the effector memory CD4+ T cells in response to IP-10 requires application of physiological levels of shear force (Fig. 3).
TNF pretreatment of the EC, which is necessary for attachment and TEM of the T cells, has been shown by antibody blocking to involve the inducible expression of adhesion molecules (18, 19). However, these results establish necessity but not sufficiency. To address the question of sufficiency, we prepared EC transductants that express levels of adhesion molecules, singly or in combination, at levels comparable to that achieved by TNF treatment (Fig. 4, A and B). Triple transductants, expressing E-selectin, ICAM-1, and VCAM-1, are fully as effective as TNF-treated monolayers at capture and stimulating IP-10-dependent TEM of effector memory CD4+ T cells (Fig. 4A) and single transductants expressing either ICAM-1 or VCAM-1 alone are nearly as effective as triple transductants (Fig. 4B). E-selectin single transductants are ineffective, presumably due to the relative scarcity of E-selectin ligand-expressing T cells in human peripheral blood. Interestingly, ICAM-1 is only effective at CD4+ T cell capture when the T cells are activated by IP-10, whereas VCAM-1 can capture T cells in the absence of exogenous chemokine.
In this report, we demonstrate three novel points relevant for human transplantation. First, IP-10, a chemokine associated with rejection, selectively acts on effector memory T cells within the resting CD4+ T cell population of peripheral blood. Second, the ability of IP-10 to induce these cells to rapidly (within 15 min) transmigrate is dependent upon the presence of shear force, a finding previously observed using SDF-1α to stimulate TEM of unfractionated CD4+ T cells. Third, the requirement for TNF activation of the endothelial cell monolayer can be wholly replaced by expression of ICAM-1 or VCAM-1.
The selective action of IP-10 on effector memory T cells coupled with the established role of IP-10 in triggering rejection would seem to implicate effector memory cells in the rejection process. Although this conclusion is concordant with studies of mouse allograft rejection (2) and our own recent report using adoptive transfer in a human skin graft model in huPBL-SCID mice (20), it must be tempered by the possibility that IP-10 also may act on other cell populations, such as CD8+ T cells or even cells other than T cells, that contribute to acute rejection. The response of IL-2-treated T cells to IP-10 further suggests that activated effector cells may also respond to this chemokine. Nevertheless, it is surprising that central memory T cells, half of which bear CXCR3 at levels comparable to effector memory cells, are unresponsive to this chemokine even though they bind to TNF-treated HUVECs to the same extent as effector memory cells (Fig. 2, A and B). The basis of IP-10 unresponsiveness remains to be elucidated. Interestingly, human memory T cells have been reported to respond to I-TAC but not IP-10 in static 4-hr TEM assays across resting HUVEC monolayers even though both of these chemokines signal through the same receptor, CXCR3 (21).
The observation that adhesion molecules are sufficient to replace TNF activation of EC for purposes of T-cell recruitment is initially surprising since TNF plays several additional roles in inflammation, including induction of chemokine expression and initiation of vascular leak (22). It is important to note that although TNF does induce several chemokines in EC, including IL-8 and MCP-1, it does not induce IP-10 in HUVEC (23). Furthermore, vascular leak is unlikely to play a significant role in our simple model of TEM. The sufficiency of VCAM-1 is consistent with its role as a mediator of both T-cell rolling and firm adhesion (9, 24). It is less expected that ICAM-1, which is not efficient at supporting either the tethering or rolling of T cells, is also sufficient to replace TNF treatment. However, in order for ICAM-1 to work, IP-10 was required, suggesting that LFA-1, unlike VLA-4, must be activated to function in our assay.
In conclusion, we have reconciled the role of IP-10 as a mediator of allograft rejection with the seeming unresponsiveness of human peripheral blood T cells to this chemokine by showing that it selectively acts on the effector memory subset without IL-2 preactivation. We have also described a simple enrichment procedure for future studies of effector memory T cell functions.
We thank Dr. Ronen Alon for helpful comments. We thank Gwendolyn Davis, Lisa Gras, and Louise Benson for preparing HUVEC cultures.
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