Evidence for involvement of lymphocyte function-associated antigen 1 in T cell migration to epidermis.

Although it is well known that in various T cell-mediated skin diseases T cells migrate preferentially to epidermis, no direct evidence has been presented in which molecules on T cells are important in directing T cell traffic to epidermis. We have previously established CD4+ autoreactive cloned T cells with a special tropism for epidermis in vitro as well as in vivo. Antibody inhibition studies demonstrated that only anti-lymphocyte function associated Ag 1 (anti-LFA-1) mAb completely inhibited the in vitro migration of the T cells toward the epidermis, whereas mAb against other T cell surface molecules had little or no effect. Monovalent F(ab) fragment of the anti-LFA-1 mAb, although less efficient, also inhibited the T cell migration. The apparent dependency of the inhibition on the anti-alpha-chain mAb suggested a major role for the alpha-chain of LFA-1 in T cell migration to epidermis. The relevance of an LFA-1-dependent mechanism to the epidermotropic migration of T cells was further strengthened by the findings that the T cell migration to epidermis was inhibited by divalent cation depletion, cytochalasin B, and low temperature. These findings indicate that the LFA-1 molecule, which is thought to be primarily involved in cell-to-cell adhesions, also plays an important role in directing T cell migration to epidermis.

Loss of epidermal integrity by T cell-mediated attack induces long term local resistance to subsequent attack. II. Thymus dependency in the induction of the resistance.

Our previous study showed that in cutaneous graft-vs-host disease (GVHD) induced by intradermal injection of autoreactive T cells the epidermal structures spontaneously recovered from the destruction became resistant to the subsequent attempts to induce the cutaneous GVHD and that the resistance correlated well with a 30-fold increase in the number of Thy-1+ epidermal cells (Thy-1+EC). We show that the resistance to the cutaneous GVHD was never induced in athymic nude mice and adult thymectomy lethal radiation and bone marrow reconstitution (ATXBM) mice under the same conditions, indicating that the induction of the resistance is dependent on the presence of thymus. A great increase in the number of Thy-1+EC was similarly observed in the epidermis of the athymic nude and ATXBM mice that spontaneously recovered from the cutaneous GVHD and that remained susceptible to the induction of the cutaneous GVHD. However, the results with B10Thy-1.1----B6 radiation chimeras clearly demonstrate that the vast majority of the increased Thy-1+EC found in the "susceptible" epidermis of the ATXBM mice were of donor bone marrow origin and there was no increase in the number of host-derived Thy-1+EC, whereas in the "resistant" epidermis of the XBM mice both Thy-1+EC populations were equally increased. The overall results indicate that the expansion of Thy-1+EC that mature in the thymus is crucial to the induction of the resistance, although the migration of bone marrow-derived Thy-1+EC precursors to the epidermis occurs quite independently of the presence of thymus.

Loss of epidermal integrity by T cell-mediated attack induces long-term local resistance to subsequent attack. I. Induction of resistance correlates with increases in Thy-1+ epidermal cell numbers.

The cutaneous graft-versus-host disease (GVHD) lesions induced by intradermal injection of cloned autoreactive T cells have been shown to subside rapidly and the epidermis returns to normal 2 wk after injection. Those mice that had spontaneously recovered from the cutaneous GVHD became resistant to subsequent attempts to induce the cutaneous GVHD by the T cells while maintaining their activity to mount delayed-type hypersensitivity (DTH) responses and to induce the enlargement of the popliteal lymph nodes (PLN). The resistance appeared to be restricted to the epidermal structures of the injection sites, suggesting the involvement of locally acting suppression mechanisms. This local resistance was not specific for the clonotype used for the induction of the resistance. A loss of the epidermal integrity by an attack of T cells capable of producing cutaneous GVHD was a prerequisite for the induction of the resistance. By up to at least 8 mo after injection of the T cells, no mice became susceptible to the cutaneous GVHD again, provided that the T cells were injected into the same footpad sites that had initially received the T cells. This resistance correlated well with the great increase (20-30-fold) in Thy-1+ EC number. The great increase in the number of Thy-1+ EC following destruction of epidermal structures may be important in protecting the epidermal integrity from an additional attack by T cells.

Both epidermal dendritic cell populations, Langerhans' cells and Thy 1+ dendritic cells are simultaneously stained by an Lyt-1 monoclonal antibody.

Both Langerhans' cells (LC) and Thy 1+ dendritic epidermal cells (DEC) are bone marrow-derived epidermal cells that are thought to play an important role in immune responses. Despite their several similarities, the surface phenotypes of both cells appear to be almost mutually exclusive. We found that an anti-Lyt-1.2 monoclonal antibody that recognizes solely an epitope on certain T cells can crossreact with a shared epitope on both cell types. The morphological details of LCs and Thy 1+ DECs stained by the anti-Lyt-1.2 MAb was superior to those stained by either anti-I-A or anti-Thy-1 MAb. The availability of this MAb enables us to simultaneously view both cell populations in the same sections.

Differential expression of lymphocyte function-associated antigen 1 (LFA-1) on epidermotropic and non-epidermotropic T-cell clones.

Various T-cell surface molecules are involved in T-cell adhesion, which is an essential requirement for epidermotropic migration of T cells. Our previous studies demonstrated that certain T-cell clones can migrate into the epidermis upon their intradermal inoculation into the footpads of recipient mice with relevant antigens, while other T-cell clones, despite their identical antigen specificities and functions, are non-epidermotropic. We therefore tested whether the differences in epidermotropism of these T cells could reside in the different levels of expression of T-cell surface molecules such as CD3, CD4, CD2, and lymphocyte function associated antigen 1 (LFA 1). The results of flow cytometric analysis showed that LFA-1 was preferentially expressed on the surface of epidermotropic T-cell clones, while non-epidermotropic T-cell clones were negative or very weakly positive for LFA-1 with one exception. After exposure to phorbol ester, epidermotropic clones with high levels of LFA-1 did not show any further up-regulation of LFA-1. In contrast, under identical conditions, significant up-regulation of LFA-1 was observed on non-epidermotropic T cells with low levels of LFA-1. However, even after exposure to phorbol ester, these T cells remained non-epidermotropic. These results suggest that the presence of high levels of LFA-1 on T cells is absolutely necessary for their epidermotropic migration, but its up-regulation is neither necessary nor sufficient to trigger the epidermotropic migration. High levels of LFA-1, regardless of cell activation, may be required to mediate stable cell adhesions leading to epidermotropic migration.

In vitro migration of L3T4+ cloned T cells to epidermis: possible role for keratinocyte-derived factors.

Three types of L3T4+ cloned T cells with different antigen specificities, auto-, allo-, and antigen-reactive, were characterized with respect to their migratory potential using an in vitro migration assay under agar gel. Autoreactive T cells, BB5, and alloreactive T cells, SK 1, both of which have been proved to be epidermotropic in vivo, showed specific directional migration to the epidermis, whereas no directional migration was seen with non-epidermotropic cloned T cells and freshly isolated lymph node T cells. Both BB5 and SK 1 cells were equally attracted to all the epidermal fragments tested regardless of their I-A antigens. The directional migration of BB5 cells to the epidermis was significantly inhibited by the co-cultivation with the epidermis, but not the dermis. Studies with cell lines, the conditioned media (CM), and recombinant interleukin (IL) 1, 2, and 3 revealed that BB5 cells were chemotactically attracted to a transformed keratinocyte cell line PAM212 and, to a lesser extent, to the CM from PAM212 and IL-2, but not to IL-1 and IL 3. These results suggest that epidermotropic T cells may be preferentially trapped in an area with a high concentration of keratinocyte-derived growth factors as well as IL-2.

Locally administered monoclonal antibodies to lymphocyte function-associated antigen 1 and to L3T4 prevent cutaneous graft-vs-host disease.

In vivo treatment with mAb directed against T cell surface molecules has been shown to prevent or reverse graft-vs-host disease (GVHD) and experimental autoimmune diseases, where T cells play a critical role. In this study we investigated the effects of in vivo administration of anti-I-A, anti-L3T4, and anti-lymphocyte function-associated Ag 1 (LFA-1) mAb on the development of murine cutaneous GVHD and delayed-type hypersensitivity responses evoked by local transfer of class II-MHC Ag-restricted, cloned autoreactive T cells. Prevention of cutaneous GVHD was achieved with local administration of either anti-L3T4 or anti-LFA-1 mAb, but not with anti-I-A mAb, while delayed-type hypersensitivity responses were inhibited by all the mAb. These results indicate that an I-A Ag-independent mechanism may be also operative in the development of the cutaneous GVHD, unlike the delayed-type hypersensitivity responses. Anti-LFA-1 mAb appeared to be more potent at inhibiting cutaneous GVHD rather than delayed-type hypersensitivity responses, whereas anti-L3T4 mAb inhibited equally both the responses. These results suggest that LFA-1 molecule may be involved in the epidermal invasion of T cells. Treatment with anti-LFA-1 mAb was found to be still, although less, effective at preventing cutaneous GVHD, even if the mAb was administered after the development of cutaneous GVHD.

In vivo effects of interferon-gamma and anti-interferon-gamma antibody on the experimentally induced lichenoid tissue reaction.

We investigated the in vivo effect of recombinant interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) treatment of mice on the development of the delayed-type hypersensitivity (DTH) reaction and lichenoid tissue reaction (LTR) following the local injection of cloned autoreactive T cells. Both the DTH reaction and the LTR were significantly enhanced by pretreatment with IFN-gamma, but not with TNF-alpha. Induction of class II MHC antigens on keratinocytes was not essential for the enhancement by IFN-gamma. Administration of anti-IFN-gamma antibody reduced the DTH reaction and LTR, although complete inhibition was not observed with our treatment regimen. The ability of IFN-gamma to increase the number of the cloned T cells invading the epidermis in vivo, is in keeping with our previous observation that IFN-gamma treatment of cultured keratinocytes markedly increased the adherence reaction between T cells and keratinocytes in vitro.

Lichenoid tissue reaction induced by local transfer of Ia-reactive T-cell clones. III. Role of Ia+ keratinocytes in the epidermotropic migration of the T cells.

The role of Ia+ keratinocytes in epidermotropism of T cells was analyzed by using self-IA specific autoreactive cloned T cells with epidermotropic nature (termed BB5) and those without it (termed C10). These T-cell clones were injected into the footpads of syngeneic nude mice whose keratinocytes had been induced to express Ia by the iv injection of normal mouse serum. Ia expression by keratinocytes was associated with the increased epidermal invasion of BB5 cells, but did not render C10 cells capable of migrating into the epidermis. The migration of the T cells to epidermis was also studied in vitro using a migration assay under agar. Ia expression by keratinocytes significantly enhanced the in vitro migration of BB5 cells to the epidermis, but had no effect on the migration of C10 cells and freshly isolated unstimulated lymphocytes. We surmise from these results that Ia+ keratinocytes may facilitate the epidermal invasion of the T cells with epidermotropic nature, but not those without it. However, the possibility certainly exists that the observed preferential migration of BB5 cells to Ia+ keratinocytes may be secondary to the alteration of other factors associated with the Ia expression. The injection of normal mouse serum was accompanied by an increase in the production of epidermal cell-derived thymocyte activating factor (ETAF) indicating that the increased ETAF production may have contributed to some of the observed preferential migration of BB5 cells.