Interleukin-17 antagonism inhibits acute but not chronic vascular rejection.

BACKGROUND: Blocking the action of interleukin (IL) 17 with an IL-17 receptor (R):Fc fusion protein inhibits T-cell proliferative responses to alloantigens and prolongs vascularized heart graft survival. In this study, we examined whether IL-17 antagonism could suppress the development of chronic rejection. METHODS: A 0.6-cm section of C57BL10 (H2b) thoracic aorta was transplanted to recipient C3H (H2k) abdominal aorta. IL-17R:Fc or control human immunoglobulin G was administered i.p. (500 microg/day) from days 0 to 6 or from days 0 to 29. Mice were killed on days 7 or 30. Grafts were examined histologically and stained for alpha-smooth muscle actin (alpha-smA). Antidonor mixed leukocyte reaction, cytotoxic T cell, and alloantibody responses were quantified. RESULTS: On day 7, control grafts showed mononuclear cell (MNC) infiltration, pronounced endothelial damage, and apoptosis of intimal and medial cell compartments. By day 30, there was concentric intimal thickening, accumulation of alpha-smA+ cells, and collagen deposition. Patchy destruction of the elastic membranes and loss of alpha-smA expression in media were evident. IL-17R:Fc for 6 days decreased MNC infiltration in the intimal and medial compartments at day 7. The endothelium was preserved (completely or partially) in all grafts. The medial compartment showed normal alpha-smA expression. Irrespective of IL-17R:Fc treatment for either 6 days or continuously, allografts harvested at day 30 showed circumferential intimal thickening, with accumulation of alpha-smA+ cells and collagen deposition. There was no effect on circulating alloantibody levels. CONCLUSIONS: These findings support a role for IL-17 in the immunopathogenesis of acute vascular rejection and demonstrate the potential of IL-17 antagonism for therapy. By contrast, IL-17 antagonism does not appear to prevent ensuing chronic graft vascular disease, in particular neointimal formation.

Microchimerism, donor dendritic cells, and alloimmune reactivity in recipients of Flt3 ligand-mobilized hemopoietic cells: modulation by tacrolimus.

Flt3 ligand (FL) is a potent hemopoietic growth factor that strikingly enhances stem cells and dendritic cells (DC) in vivo. We examined the impact of infusing FL-mobilized bone marrow (BM) cells on microchimerism and anti-donor reactivity in normal and tacrolimus-immunosuppressed, noncytoablated allogeneic recipients. BM from B10 (H2b) mice given FL (10 microg/day; days 0-8; FL-BM) contained a 7-fold higher incidence of potentially tolerogenic immature CD11c+ DC (CD40low, CD80low, CD86low, MHC IIlow) that induced alloantigen-specific T cell hyporesponsiveness in vitro. C3H (H2k) mice received 50 x 106 normal or FL-BM cells (day 0) and tacrolimus (2 mg/kg/day; days 0-12). On day 15, enhanced numbers of donor (IAb+) cells were detected in the thymi and spleens of FL-BM recipients. Tacrolimus markedly enhanced microchimerism, which declined as a function of time. Ex vivo splenocyte proliferative and CTL responses and Th1 cytokine (IFN-gamma) production in response to donor alloantigens were augmented by FL-BM infusion, but reduced by tacrolimus. Systemic infusion of purified FL-BM immature DC, equivalent in number to that in corresponding whole BM, confirmed their capacity to sensitize, rather than tolerize, recipient T cells in vivo. In vitro, tacrolimus suppressed GM-CSF-stimulated growth of myeloid DC from normal BM much more effectively than from FL-BM without affecting MHC class II or costimulatory molecule expression. Infusion of normal B10 BM cells at the time of transplant prolonged C3H heart allograft survival, whereas FL-BM cells did not. A therapeutic effect of tacrolimus on graft survival was observed in combination with normal, but not FL-BM cells. These findings suggest the need for alternative immunosuppressive strategies to calcineurin inhibition to enable the engraftment, survival, and immunomodulatory function of FL-enhanced, immature donor DC.

Flt3 ligand (FL) and its influence on immune reactivity.

Flt3 (fms-like tyrosine kinase 3) ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of progenitor and stem cells both in vivo and in vitro. Its capacity to augment strikingly the numbers of dendritic cells (rare antigen-presenting cells that induce and regulate immune responses) in mice and humans has stimulated considerable interest in its value as an investigational tool and therapeutic agent. In this review, we survey the hematopoietic properties and immunobiology of FL, and examine its therapeutic potential.

Evidence for a role of IL-17 in organ allograft rejection: IL-17 promotes the functional differentiation of dendritic cell progenitors.

IL-17 is a T cell-derived cytokine that stimulates stromal cells and macrophages to secrete proinflammatory cytokines. We hypothesized that IL-17 might play a role in alloimmune responses, and that interference with its activity might suppress allograft rejection. IL-17R:Fc or control IgG was added at the start of mouse MLR or was administered i.p. (100-500 microg/day) for different durations post-transplant to murine recipients of MHC-mismatched cardiac allografts. IL-17R:Fc (50-200 microg/ml) markedly inhibited T cell proliferation in vitro and significantly prolonged nonvascularized cardiac allograft median survival time from 13 to 20 days (100 microg/day; days 0 and 1) or to 19 days (100-300 microg/day; days 0-4). Survival of vascularized grafts was also extended significantly from 10.5 to 19 days by IL-17R:Fc (500 microg/day; days 0-6). To address a possible mechanism by which IL-17 may promote alloreactivity, we examined the influence of IL-17 on the differentiation and function of bone marrow-derived cells propagated in granulocyte-macrophage CSF with or without IL-4 to promote dendritic cell (DC) growth. A minor proportion of CD11c+ DC expressed the IL-17R. IL-17 promoted the maturation of DC progenitors, as evidenced by increased cell surface expression of CD11c, costimulatory molecules (CD40, CD80, CD86), and MHC class II Ag, and allostimulatory capacity. IL-17 had a lesser effect on the phenotype and function of more fully differentiated myeloid DC. These findings suggest a role for IL-17 in allogeneic T cell proliferation that may be mediated in part via a maturation-inducing effect on DC. IL-17 appears to be a novel target for therapeutic intervention in allograft rejection.

Flt-3 ligand increases microchimerism but can prevent the therapeutic effect of donor bone marrow in transiently immunosuppressed cardiac allograft recipients.

C3H (H2k) mice received 50 x 10(6) B10 (H2b) bone marrow (BM) cells either alone or with flt-3 ligand (FL) (10 microg/day), tacrolimus (2 mg/kg/day), or both agents for 7 days. Donor MHC class II+ (IAb+) cells were quantitated in spleens by immunohistochemical analysis, and donor class II DNA detected in BM by PCR. Donor cells were rare in the BM alone and BM + FL groups, whereas there was a substantial increase in chimerism in the BM + tacrolimus group. Addition of FL to BM + tacrolimus led to a further eightfold increase in donor cells and enhanced donor DNA compared with the BM + tacrolimus group. This increase in donor cells was almost 500-fold compared with BM alone. C3H recipients of B10 heart allografts given perioperative B10 BM and tacrolimus (days 0-13) exhibited a markedly extended median graft survival time (MST, 42 days) compared with those given tacrolimus alone (MST, 22 days). Addition of FL (10 microg/day; 7 days) to BM + tacrolimus prevented the beneficial effect of donor BM (MST, 18 days). BM alone or BM + FL resulted in uniform early heart graft failure (MST < 8 days). Functional studies revealed maximal antidonor MLR and CTL activities in the BM- and BM + FL-treated groups, with minimal activity in the tacrolimus-treated groups. Thus, dramatic growth factor-induced increases in chimerism achieved under cover of immunosuppression may result in augmented antidonor T cell reactivity and reduced graft survival after immunosuppressive drug withdrawal. With FL, this may reflect striking augmentation of immunostimulatory dendritic cells.

Donor bone marrow potentiates the effect of tacrolimus on nonvascularized heart allograft survival: association with microchimerism and growth of donor dendritic cell progenitors from recipient bone marrow.

BACKGROUND: The influence of donor hematopoietic cell microchimerism on organ allograft survival has been studied largely in vascularized transplant models. Here, we examine the impact of donor bone marrow (BM) cells administered intravenously together with transient systemic tacrolimus therapy on microchimerism, the survival of nonvascularized cardiac allografts, and growth of donor antigen-presenting cells [dendritic cells (DCs)] from recipient BM. METHODS: Adult male C3H (H2k) mice received heterotopic heart transplants from B10 (H2b) donors in the dorsal ear pinna. They were given no further treatment, or either a short course of tacrolimus (FK506; 2 mg/kg i.p. from day 0 to day 13), unmodified donor BM cells (50x10(6) i.v. on day 0) or both treatments. Grafts were examined daily for contractile activity. Anti-donor cytotoxic T lymphocyte responses were determined in recipients' spleens. Microchimerism (IAb+ cells) was demonstrated by immunocytochemical staining of spleens, and of cells expanded from recipient BM using cytokines and culture conditions that promote the growth of DCs. RESULTS: Tacrolimus alone significantly prolonged median heart graft survival time from 10 to 22 days (P<0.001). BM alone failed to prolong graft survival. By contrast, tacrolimus + donor BM resulted in a mean survival time of 42 days (P<0.01 compared with tacrolimus treatment alone). This marked increase in heart allograft survival was associated with reduced anti-donor cytotoxic T lymphocyte responses attributable to a nonspecific effect of tacrolimus. In addition, however, a link was observed between the beneficial effect of donor BM and comparatively large numbers of donor major histocompatibility complex class II (IAb+)-positive cells in recipients' spleens, and in cultures of granulocyte-macrophage colony-stimulating factor + interleukin-4-stimulated DCs from recipients' BM. No donor-derived cells were propagated from heart graft recipients given either tacrolimus or donor BM alone. CONCLUSIONS: This nonvascularized organ transplant model demonstrates the positive effect on allograft survival of donor BM given at the time of transplant to transiently immunosuppressed recipients. The findings also reveal links between hematopoietic cell chimerism, the presence of donor DC progenitors in recipient BM, and organ allograft survival.

Impact of Flt-3 ligand on donor-derived antigen presenting cells and alloimmune reactivity in heart graft recipients given adjuvant donor bone marrow.

The influence of the haematopoietic growth factor Flt-3 ligand (FL) on the incidence and function of donor major histocompatibility complex (MHC) class II+ cells in the lymphoid tissues of noncytoablated recipients of heart allografts and donor bone marrow (BM) cells was investigated. C3H (H2k) mice received a nonvascularized B10 (H2b) heart allograft in the dorsal ear pinna, followed by an i.v. infusion of 50 x 10(6) donor BM cells. They were given FL (10 microg/day i.p., x7 days), tacrolimus (2mg/kg/day i.p., x13 days) or both agents immediately following heart transplantation (HTx) and were killed 10 or 21 days later. Their BM cells were propagated in vitro in granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 for 5 days to promote the growth of dendritic cells (DC). Donor DC were identified by immunocytochemical staining. Spleens were harvested, and donor (IAb+) cells enumerated by immunohistochemical analysis. Donor MHC class II DNA was detected in spleens and cultured BM-derived cells by reverse transcriptase-polymerase chain reaction (RT-PCR). A striking increase in donor MHC class II+ cells was noted in both the spleen and BM of the BM + tacrolimus-treated group compared to either the BM alone, or BM + FL-treated groups. Addition of FL treatment to BM + tacrolimus led to a further increase in donor cells in spleen (three-fold at 10 days, and two-fold at 21 days). The increase in donor cells at 10 days was almost 140-fold compared to that with donor BM alone. PCR analysis at this time revealed enhanced donor DNA in the BM + FL + tacrolimus group compared to that in the BM + tacrolimus group. FL treatment augmented mixed leucocyte reactions (MLR) and cytotoxic T lymphocyte (CTL) activity of host spleen cells against donor alloantigens. These effects were reversed by tacrolimus administration. Histopathology of heart grafts from tacrolimus-treated animals at 10 and 21 days showed absence or substantial reduction in cellular infiltration, and the preservation of viable myocardium. By contrast, in untreated mice, or animals given BM or BM + FL alone, there was marked cellular infiltration, and features of accelerated rejection. Donor-derived DC could be propagated in vitro from the BM of heart transplant recipients given donor BM, especially from mice that also received tacrolimus +/- FL. At day 21, donor-derived cells could only be propagated from the BM + FL + tacrolimus-treated group. These findings show that numbers of donor antigen presenting cells (APC) or their progenitors can be markedly increased in conventionally immunosuppressed organ allograft recipients given donor BM + a potent haematopoietic and DC-growth promoting cytokine. Although withdrawal of systemic immunosuppression appears to allow exhibition of the potential allostimulatory activity of these donor APC leading to rejection, the model provides a useful basis for further evaluation of the persistence and manipulation of donor haematopoietic cells and in particular, donor-derived APC, on the outcome of organ transplantation.

Striking augmentation of hematopoietic cell chimerism in noncytoablated allogeneic bone marrow recipients by FLT3 ligand and tacrolimus.

The influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and the recently identified hematopoietic stem-progenitor cell mobilizing factor flt3 ligand (FL) on donor leukocyte microchimerism in noncytodepleted recipients of allogeneic bone marrow (BM) was compared. B10 mice (H2b) given 50x10(6) allogeneic (B10.BR [H2k]) BM cells also received either GM-CSF (4 microg/day s.c.), FL (10 microg/day i.p.), or no cytokine, with or without concomitant tacrolimus (formerly FK506; 2 mg/kg) from day 0. Chimerism was quantitated in the spleen 7 days after transplantation by both polymerase chain reaction (donor DNA [major histocompatibility complex class II; I-E(k)]) and immunohistochemical (donor [I-E(k)+] cell) analyses. Whereas GM-CSF alone significantly augmented (fivefold) the level of donor DNA in recipients' spleens, FL alone caused a significant (60%) reduction. Donor DNA was increased 10-fold by tacrolimus alone, whereas coadministration of GM-CSF and tacrolimus resulted in a greater than additive effect (28-fold increase). A much more striking effect was observed with FL + tacrolimus (>125-fold increase in donor DNA compared with BM alone). These findings were reflected in the relative numbers of donor major histocompatibility complex class II+ cells (many resembling dendritic cells) detected in spleens, although quantitative differences among the groups were less pronounced. Evaluation of cytotoxic T lymphocyte generation by BM recipients' spleen cells revealed that FL alone augmented antidonor immunity and that this was reversed by tacrolimus. Thus, although FL may potentiate antidonor reactivity in nonimmunosuppressed, allogeneic BM recipients, it exhibits potent chimerism-enhancing activity when coadministered with recipient immunosuppressive therapy.

Adenosine acts as an endogenous modulator of IL-2-dependent proliferation of cytotoxic T lymphocytes.

CTLL-2 cells are a clone of CTL that are dependent on IL-2 for proliferation. In addition to various cytokine receptors, we observed that these cells express three subtypes of adenosine receptors (ARs). In an initial attempt to delineate the functions of these receptors in CTLL-2 cells, we tested their role in proliferation. Elimination of endogenous adenosine with adenosine deaminase (ADA) markedly suppressed IL-2-dependent proliferation of these cells. This proliferative response was restored by addition of R-phenylisopropyladenosine (R-PIA), a non-hydrolyzable adenosine analogue. The stimulatory response to R-PIA was attenuated following blockade of ARs by 0.5 mM theophylline and 10 microM BW-A1433, but not by blockade of the A1AR with 100 nM xanthine amine congener. The rank order of potency of adenosine analogues in proliferation assays was R-PIA > or = N-ethylcarboxamide adenosine > S-PIA > PAPA-APEC (a substituted ethylamino-5'-N-ethylcarboxamidoadenosine). These data suggest a potential role of the A3AR in the proliferative response. R-PIA stimulates production of 1,4,5-inositol trisphosphate in CTLL-2 cells, suggesting a role of the phospholipase C signaling pathway in the proliferative response. A23187 (100 nM) and phorbol 12,13 dibutyrate (10 nM), but not 4 alpha-phorbol (10 nM), were able to restore IL-2-dependent CTLL-2 proliferation in the presence of ADA. Furthermore, inhibition of protein kinase C by staurosporine (10 nM) and of phospholipase C by tricyclodecan-9-yl-xanthogenate (D609) blocked R-PIA-mediated cell proliferation. These data demonstrate an obligatory role of adenosine in IL-2-dependent proliferation of CTLL-2 cells and support the involvement of an AR-stimulated phospholipase C signaling pathway in this process.