Competitive homing assays to study gut-tropic t cell migration.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressions) displayed on endothelial cells (1 2). Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment. Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Selective requirements for leukocyte adhesion molecules in models of acute and chronic cutaneous inflammation: participation of E- and P- but not L-selectin.

Adhesion molecules borne by both endothelial cells and circulating leukocytes are in large measure responsible for guiding the process of extravasation. The selectin family has been primarily associated with the early stages of adhesion involving initial contact and rolling. A significant body of evidence has accumulated indicating a fundamental role for the endothelial members of this family, E- and P-selectin, in a variety of inflammatory states and models. Although originally identified as the lymph node-specific lymphocyte homing receptor, L-selectin has also been suggested to play an important role in leukocyte recruitment to sites of inflammation. We have recently demonstrated, using L-selectin-deficient mice, that defects in contact hypersensitivity (CHS) responses are in essence due to the inability of T cells to home to and be sensitized within peripheral lymph nodes, whereas nonspecific effector cells are fully capable of entry into sites of cutaneous inflammation (Catalina et al, J Exp Med 184:2341, 1996). In the present study, we perform an analysis of adhesion molecule usage in two models of skin inflammation and show in both L-selectin-deficient as well as wild-type mice that a combination of P- and E-selectin is crucial for the development of both acute (croton oil) and chronic (contact hypersensitivity) inflammation at sites of the skin, whereas L-selectin does not appear to play a significant role. Moreover, alpha4 integrins are shown to be integral to a CHS but not an acute irritant response, whereas CD44 does not significantly contribute to either. These results provide a systematic examination in one study of major adhesion molecules that are critical in acute and chronic skin inflammation. They reinforce the essential role of the collaboration of E- and P-selectin in both specific and nonspecific skin inflammatory responses and the importance of alpha4 in the specific response only. In addition, they substantiate only a limited role, if any, for L-selectin in these cutaneous effector mechanisms and demonstrate the essential equivalence in this analysis of L-selectin-deficient mice compared with normal mice treated with blocking antibodies.

Bromelain protease F9 reduces the CD44 mediated adhesion of human peripheral blood lymphocytes to human umbilical vein endothelial cells.

The thiol protease bromelain has been shown to remove T-cell CD44 molecules from lymphocytes and to affect T-cell activation. We investigated the effect of a highly purified bromelain protease F9 (F9) on the adhesion of peripheral blood lymphocytes (PBL) to human umbilical vein endothelial cells (HUVEC). Preincubation of the lymphocytes with F9 reduced the adherence to about 20% of unstimulated and to about 30% of phorbol-dibutyrate (P(Bu)2) stimulated lymphocytes. Using flow cytometry, both crude bromelain and protease F9 reduced the expression of CD44, but not of LFA-1, on PBL. F9 was about 10 times more active than crude bromelain; at 2.5 micrograms/ml of F9 about 97% inhibition of CD44 expression was found. A mAb against CD44 was tested and found to block the F9-induced decrease in PBL-binding to HUVEC. The results indicate that F9 selectively decreases the CD44 mediated binding of PBL to HUVEC.

Reentry of T cells to the adult thymus is restricted to activated T cells.

To seek information on the capacity of mature T cells to migrate to the thymus, mice were injected with Thy-1-marked populations enriched for resting T cells or T blast cells; localization of the donor cells in the host thymus was assessed by staining cryostat sections of thymus and by FACS analysis of cell suspensions. With injection of purified resting T cells, thymic homing was extremely limited, even with injection of large doses of cells. By contrast, in vivo generated T blast cells migrated to the thymus in substantial numbers. Thymic homing by T blasts was greater than 50-fold more efficient than with resting T cells. Blast cells localized largely in the medulla and remained in the thymus for at least 1 mo post-transfer. Interestingly, localization of T blasts in the thymus was 10-fold higher in irradiated hosts than normal hosts. Thymic homing was especially prominent in mice injected with T blasts incubated in vitro with the DNA precursor, 125I-5-iodo-2'deoxyuridine (125IDUR); with transfer of 125IDUR-labeled blasts to irradiated hosts, up to 5% of the injected counts localized in the host thymus. These data suggest that thymic homing by T blasts might be largely restricted to cells in S phase. The physiological significance of blast cell entry to the thymus is unclear. The possibility that these cells participate in intrathymic tolerance induction is discussed.