This study demonstrates that egression of human T cells from the lung across bronchial epithelium is a multistep process. In particular, we describe an LFA-1-dependent event during which T cells bind ICAM-1 and ICAM-2 on the basolateral epithelium, before crossing the junctions. We present evidence that the ICAM-1 and ICAM-2, together with a chemoattractant, direct the T cells to the lateral interepithelial cell space. When LFA-1 is blocked, the T cells move over the basal epithelium but do not appear to recognize the intercellular junctions and therefore do not cross them. In addition, we show an essential role of epithelial CAR for successful transepithelial migration.
Although transepithelial migration is inhibited when ICAM-1 on the basal epithelium is blocked, the inhibition is less than when LFA-1 on the T cells is blocked, suggesting other ligands for LFA-1. ICAM-2 and ICAM-3 were thought to be restricted to endothelial and hematopoietic cells, but RNA for ICAM-2 has been shown in human bronchial epithelium (24)
. Here, we demonstrate that both primary and immortalized human airway cells express ICAM-2. Moreover, we show that inhibition of both ICAM-1 and ICAM-2 has the same effect as inhibition of LFA-1, demonstrating, for the first time, a functional role for ICAM-2 in the transepithelial migration of leukocytes. These findings highlight a completely new role for ICAM-2 as a molecule involved in the resolution of inflammation and may explain the paradoxical findings of worsening asthma in ICAM-2-deficient mice (2)
. Our findings also emphasize the potential importance of differential tissue regulation of molecules, such as ICAM-1 and ICAM-2, which are involved in both the recruitment and clearance of leukocytes from epithelial organs.
Interestingly, blocking mAbs against ICAM-1 or ICAM-2 are only effective when placed on the basal side of the intercellular junctions, implying that, although LFA-1/ICAM-1 and ICAM-2 interactions are not required for adhesion to the basal surface of the epithelium, they are required in a postadhesion step prior to diapedesis. There are technical difficulties in the study of egression: T cells migrate across the epithelium in a basal to apical direction, and there are optical limitations imposed by imaging through the monolayer (9)
. For this reason, we have used confocal microscopy to visualize and quantify T-cell adhesion to the basal epithelium prior to transepithelial migration. We show that when LFA-1 is blocked, the T cells are still able to adhere, polarize, and elongate along the epithelium, but they are elongated and appear to cross several intercellular junctions, without migrating across them. So that although egressing T cells do not require ICAM-1/2 for adhesion to the basal epithelium, they do require the limited amount of ICAM-1/2 at the interepithelial space to guide them.
The LFA-1-dependent postadhesion step, whereby T cells move into the subjunctional epithelial intercellular space, resembles locomotion (16, 17)
. Schenkel reported that inhibition of monocyte integrin CD18 during transendothelial migration resulted in pirouetting cells, unable to recognize ligand on the apical endothelium; in contrast, inhibition of endothelial ICAM-1 and ICAM-2 resulted in elongated monocytes crossing many junctions on the apical endothelium, leading him to conclude that there were other unidentified endothelial ligands for CD18 (17)
. In contrast, in our epithelial assay, inhibition of LFA-1 (the only CD18 integrin expressed on these T cells) did not prevent lymphocytes becoming polarized and elongated. This suggests that integrin/ligand pairings other than LFA-1/ICAM-1 and ICAM-2 are involved in adhesion and migration over the basal epithelium (which is unsurprising given the relative paucity of ICAM-1/2 on the basal epithelium compared to the apical endothelium), but that LFA-1/ICAM-1 and ICAM-2 are required to guide T cells into the lateral intercellular space so that they can contact the tight junction. The dramatic effect on egression seen when blocking either ICAM-1 or ICAM-2, but leaving the other ligand functional suggests that even subtle disruptions of the ICAM-1/2 gradient may prevent egression. This may be particularly important in the epithelium where the cells are tall and columnar; endothelial cells are flatter and the tight junctions between them may be relatively more accessible.
We show that CAR is important for the movement of T cells across the bronchial epithelium when tight junctions are intact. CAR is a tight junction protein, which is used by luminal coxsackie and adenoviruses to enter the lung (25)
or by basal viruses to disrupt tight junctions (26)
. The normal physiological function of CAR was unclear until it was recently shown to be involved in neutrophil migration across the colonic epithelium (27)
. The inhibition of T-cell egression seen when CAR is blocked in the bronchial epithelium can be overcome when the epithelial junctions are disrupted by pretreatment of the epithelium with cytochalasin D. Under these conditions, mAbs that block LFA-1 are still able to inhibit transepithelial migration. This is evidence that the LFA-1-dependent step is distinct from diapedesis and takes place before it. The finding that blocking CAR only partially inhibits egression suggests that other CAR-independent pathways may be involved. Leukocytes migrate across endothelial monolayers via
both transcellular and paracellular passages (28
and it is possible that similar pathways exist across an epithelial monolayer.
The surprising observation that, in response to a given concentration of CXCL11, a similar number of T cells migrate across a filter as migrate across a tight epithelial monolayer, suggests that the epithelium plays an active part in the migration. In examining the role of Rho, Rac, and Cdc42 GTPases in the bronchial epithelial cells, we find that ICAM-1 cross-linking does not induce junctional rearrangements or permeability changes, but inhibition of Rho-kinase causes partial disruption of the tight junctions and an increase in transepithelial migration, similar to that seen with cytochalasin D treatment. A novel finding is that wiskostatin, an inhibitor of the Cdc42 effector N-WASp, disrupts the epithelial cell junctions but paradoxically causes an inhibition of transepithelial migration. N-WASp is a signaling molecule that, when bound by active Cdc42 is able to initiate Arp2/3-dependent cortical actin polymerization (32)
, including that involved in the formation of filopodia, lamellipodia and tight junctions, membrane trafficking, and the intracytoplasmic movement of various pathogens [reviewed by Takenawa and Suetsugu (33)
Because inhibition of epithelial N-WASp and inhibition of lymphocyte LFA-1 appear to inhibit T cells at the same stage in their egression, we examined the effect of wiskostatin on epithelial ICAM-1 distribution. We find a redistribution of ICAM-1 with disruption of the normal basal-to-lateral gradient. This suggests that the effect of wiskostatin may be due, in part, to disruption of the haptotactic gradient of ICAMs. Cytochalasin D disrupts tight junctions to the same extent as wiskostatin, but it has a different effect on ICAM-1 distribution, suggesting that the effect of wiskostatin on ICAM-1 is not wholly attributable to junctional disruption and a resultant lateral mobility of membrane proteins.
After successful egression, T cells down-regulate LFA-1 on arrival in the airway (34)
, allowing the cells to detach from the epithelial cell surface and be removed on the mucociliary escalator (35)
. An increase in the detachment rate probably accounts for the apparent increase in transepithelial migration seen when the mAb against ICAM-1 is presented apically. These findings emphasize the importance not just of overall levels of adhesion molecules but also of their distribution.
Although our study investigates the egression of human T cells, we believe that these results may extend to other leukocytes. The advantages of our system are that we use an exclusive T-cell chemoattractant that we have demonstrated to be produced in a polarized manner by the bronchial epithelium in health and disease, and that does not disrupt the monolayer or alter adhesion molecule expression and distribution (9)
. In addition, in contrast to myeloid cells, primary human T cells express no or very low levels of the promiscuous β2 integrin MAC-1 (αMβ2;CD18:CD11b), and the results involving LFA-1/ICAM-1 are therefore easier to interpret (36)
. The use of lymphocytes also avoids the artifactual disruption of endothelial and epithelial monolayers by granulocytic cells, which has been described [reviewed by Zen and Parkos (37)
In conclusion, we have identified key steps in the CXCL11-induced egression of T cells across a bronchial epithelial monolayer and have shown that the egression is a multistep process, analogous to that across the endothelium. We describe an essential postadhesion step that requires LFA-1 binding to ICAM-1/2 expressed on the lateral surface of the epithelial cells, basal to the tight junctions. This interaction results in a movement of T cells into the lateral intercellular space and positioned for subsequent diapedesis. The T cells then interact with CAR and probably other junctional proteins to allow movement across the tight junctions. The T cells finally deadhere from the apical ICAMs, and the released cells are eventually cleared from the airway by expectoration.