The present study demonstrates that repeated HGF gene transfection into BDF1 mice prevents the development of chronic GVHD induced by the injection of DBA/2 spleen cells. HGF transfection significantly inhibited proteinuria and histopathological changes of the kidneys, liver and salivary glands caused by chronic GVHD.
The parent-to-F1 murine model of chronic GVHD exhibits Th2-mediated immune responses, such as polyclonal B cell activation, autoantibody formation, and decreased CTL responses, that closely resemble lupus-like autoimmune disease. Rus and colleagues [5
] reported that Th2 cytokine secretion and B cell activation may be early events in both acute and chronic GVHD. It is thought that the transition to chronic GVHD involves the failure of CD8+ anti-host CTLs to kill activated host B cells [5
]. In this context, therapeutic strategies tested in chronic GVHD mice have included the use of Th1-inducing cytokines [13
]. Early treatment with IL-12 (on days 0 to 4 after induction of chronic GVHD) but not late treatment (on days 8 to 12 after induction of chronic GVHD) generated anti-host CTLs that eliminated host autoantibody-producing B cells, thereby converting chronic GVHD into acute GVHD [13
]. However, regardless of treatment schedule (treatment on days 0 to 5 or days 8 to 13 after induction of chronic GVHD), IL-18 treatment generated anti-host CTLs, but did not induce acute GVHD [14
In contrast to Th1-inducing cytokine treatment, HGF did not generate anti-host CTLs in vivo
. Although Th1-inducing cytokines induced the development of DBA/2-derived Th1 in a DBA/2 anti-BDF1 MLR [25
], the addition of HGF did not induce Th1 in vitro
(data not shown). However, HGF treatment inhibited IL-4 mRNA expression in chronic GVHD target organs, splenic B cell expansion, and autoantibody production in chronic GVHD mice. Thus, it appears that HGF inhibited lupus-like autoimmune disease through the inhibition of Th2 generation rather than by induction of Th1.
The precise mechanisms by which HGF inhibits Th2-mediated responses in chronic GVHD mice remain unclear. Possible mechanisms include that HGF suppresses MHC class II expression by host B cells, leading to reduced antigen presentation to donor CD4+ T cells. Indeed, we observed decreased MHC class II expression on host B cells from HGF-treated chronic GVHD mice. While the addition of HGF to an in vitro DBA/2 anti-BDF1 MLR inhibited the capacity of BDF1 B cells to increase MHC class II expression, HGF did not inhibit IL-4-induced B cell MHC class II expression, which suggested that HGF did not directly suppress MHC class II expression on host B cells.
DCs play a pivotal role in determining the balance between responsiveness and tolerance in the immune system [26
], and the persistence of host DCs following bone marrow transplantation is correlated with the development of severe acute and chronic GVHD [28
]. Chronic DC activation, such as by CD40L over-expression in basal epidermal layers that accelerates DC maturation, leads to autoimmunity [30
]. Immature myeloid DCs are loaded with self-antigen-derived molecules and, upon interaction with T cells, deliver a signal that results in T cell deletion and anergy [31
]. Modified myeloid DCs can act as regulatory DCs to protect against acute GVHD [32
]. We observed that in a DBA/2 anti-BDF1 MLR, which contains DBA/2 CD4+ T cells and BDF1 DCs, the addition of HGF significantly inhibited the generation of DBA/2 Th2. We also observed that c-Met/HGF receptors are expressed by both DCs and CD4+ T cells (data not shown) and that HGF significantly down-regulated CD28 expression on DBA/2 CD4+ T cells stimulated by BDF1 DCs. We also observed down-regulation of CD86 expression on BDF1 DCs cultured with DBA/2 CD4+ T cells. HGF may act on the CD28-CD86 pathway, thereby inhibiting Th2 generation.
We recently demonstrated that repeated transfection of the human HGF gene into skeletal muscle in a bone marrow transplantation model of GVHD promoted hematopoietic function and strongly inhibited acute GVHD by limiting tissue damage and the subsequent endotoxin-mediated inflammatory cascade [15
]. The principal mechanisms by which HGF blocks acute GVHD appeared to involve the protection of target organs from injury through anti-apoptotic effects and the inhibition of subsequent inflammatory cytokine reactions [16
]. In the present study, we demonstrated that HGF inhibited the increased Th2-mediated immune response in chronic GVHD mice. Thus, HGF treatment may be beneficial for both Th1-mediated acute GVHD and Th2-mediated autoimmune chronic GVHD.