Using models of human T cell responses to allogeneic vascular cells in vitro and in vivo, we demonstrate important roles for graft-derived IL-1 in shaping the host antigraft adaptive immune response and in promoting T cell–mediated injury to allogeneic vasculature. Specifically, we demonstrate that the EC lining of human artery segments interposed into the infrarenal aortae of immunodeficient CB.17 SCID/beige mice expresses IL-1α and that blockade of IL-1 with IL-1Ra reduces T cell recruitment to the graft intima. Thus, blockade of IL-1 appears to protect allograft arteries from a process that resembles acute cell-mediated vascular rejection, sometimes described as “intimal arteritis.”
The use of human materials in our work allows for two important advances beyond the insights gained from rodent transplant models. First, we focus on alloreactive memory cells, which are present in significant numbers in clinical transplant recipients but are typically lacking in rodents. Second, we can identify signals that alter human T cell responses (e.g., IL-17 production), which can differ significantly from those that act in mice. Translational studies using humanized mouse models, despite being limited by scarce materials and few tools for genetic manipulation, are likely to more accurately predict potentially useful therapies (43
). Our study contributes several novel observations regarding the interactions of IL-1 and IL-17: (a) IL-1α localized on ECs can selectively enhance IL-17 production from human alloreactive memory CD4+
T cells, especially upon restimulation; (b) allograft-expressed IL-1 promotes IL-17 production from human T cells in vivo; (c) IL-17 drives inflammatory responses and inflammatory T cell recruitment during T cell–mediated human allograft rejection in vivo; and (d) the principle vascular cell target of IL-17 appears to be the SMCs rather than ECs, a striking difference from the inflammatory responses mediated by TNF or IL-1.
Although best known as an inducer of acute inflammation, IL-1 can also modulate adaptive immune responses. Recent studies have demonstrated a critical role for IL-1 in inducing Th17 differentiation in human naive T cells (31
). IL-1 can also participate in the differentiation of mouse Th17 cells (28
); however, some effects of IL-1 on mouse T cell differentiation appear to differ between mouse strains (45
). Although most studies have focused on differentiation of naive T cells, cytokine production from memory CD4+
T cells can also be modulated by the environment. For example, polarized Th1 cells from both humans and mice can be induced to produce Th2 cytokines by activation under strongly Th2-polarizing conditions and vice versa, although this plasticity may be more pronounced in human compared with mouse T cells (46
). In addition, we have previously shown that addition of exogenous IL-1 to human EC–T cell co-cultures can selectively skew alloreactive memory CD4+
T cells toward IL-17 production (12
). We now show that human ECs express sufficient bioavailable IL-1α, up-regulated either by treatment with exogenous TNF or by TNF produced by alloreactive memory CD4+
T cells, to selectively promote IL-17 production from alloreactive memory T cells. The presence of this cell-associated, bioactive IL-1α suggests that serum levels of IL-1, even when collected from a local site, may not accurately predict the activity of this mediator in modulating T cell–driven pathological processes.
Although several cytokines, including IL-1, IL-6, IL-23, TGF-β, and IL-21, have been suggested to contribute to the development of IL-17–producing T cells in vitro (12
), little information exists on which factors may be relevant in human immune responses in vivo (50
). Using a human–mouse model of human artery allograft rejection, we provide the first evidence that IL-1 promotes IL-17 production from human T cells in vivo. Blockade of IL-1 reduces IL-17 expression but increases IFN-γ expression in rejecting human artery allografts, suggesting that IL-1 promotes T cell production of IL-17 at the expense of IFN-γ in vivo. This change in cytokine expression could reflect a role for IL-1 in directing selective T cell recruitment or T cell differentiation in the periphery; however, we did not observe significant alterations in the expression of several genes that are preferentially expressed by human IL-17–producing T cells (CCR6, IL-23R, and RORC) with IL-1 blockade in artery grafts in vivo or in purified memory CD4+
T cells activated with ECs expressing high levels of IL-1α in vitro. These observations suggest that, rather than having an effect on lineage specification, IL-1 may alter effector cytokine production in undifferentiated or partially differentiated human memory T cells, which show considerable plasticity in cytokine production phenotypes (47
). This possibility may be particularly relevant for IL-17, which can be produced by T cells that also make IFN-γ (25
). Indeed, human IL-17–producing and IFN-γ–producing T cell clones show significant overlap in expression of differentiation markers and have been suggested to share a common developmental program (26
). Nevertheless, the data presented in this paper suggest that the effector responses of alloreactive memory T cells, in this case production of IL-17, can be modulated by signals in the microenvironment of the target tissue (e.g., by IL-1α expressed on graft endothelium).
We also observed that in addition to decreasing IL-17 expression by artery-infiltrating T cells, IL-1 blockade significantly diminished T cell–mediated graft injury, as assessed by T cell intimal infiltration, neointimal expansion, and outward arterial remodeling. Our group has previously demonstrated a key role for T cell production of IFN-γ in promoting arterial injury and pathological remodeling in this model (51
); therefore, it may seem initially surprising that IL-1 blockade reduces neointimal expansion without reducing IFN-γ expression by those T cells that do infiltrate the vessel wall. The likely explanation is that the effect of IL-1 blockade on the extent of T cell infiltration into the neointima significantly reduces the total amount of IFN-γ in the intima. The effect on intimal T cell numbers by IL-1 blockade may relate to T cell recruitment, T cell retention, or T cell proliferation and survival, all of which could be influenced by IL-1. The failure to reduce IFN-γ by individual T cells in vivo differs from our observed effects in vitro, when T cells were co-cultured with allogeneic ECs. These differences may relate to an incomplete blockade of IL-1 by IL-1Ra in vivo, perhaps because of rapid clearance, or may suggest that alloreactive T cells within the wall more closely resemble our secondary stimulation of T cells in which the effects of IL-1 on IFN-γ during the initial activation are no longer observed.
The correlation between reduced intimal infiltration and reduced IL-17 expression caused by IL-1 blockade suggested a potential pathological role for IL-17; however, unlike IL-1 blockade, IL-17 neutralization failed to reduce T cell intimal infiltration and neointimal expansion. As IL-1 appears to be an upstream inducer of IL-17, it is possible that blockade of IL-1 may inhibit production of other Th17-derived cytokines, including IL-17F and IL-22, and may thus be more effective than blockade of individual Th17 effector cytokines in limiting Th17-mediated pathology. We were unable to evaluate this possibility because the expression levels of IL-17F and IL-22 were too low to accurately measure in these experiments. Alternatively, our inability to detect IL-17F and IL-22 may suggest that the IL-17A detected in the artery grafts is produced by undifferentiated memory T cells or by IFN-γ/IL-17 double producers rather than bona fide Th17 cells. It is also clear that IL-1 blockade has other protective effects beyond modulating T cell cytokine profiles, such as reducing the extent of T cell infiltration.
Elevated levels of IL-17 have been reported in several human autoimmune diseases; however, specific effects of human T cell production of IL-17 in vivo have yet to be reported. In the model of human artery graft rejection described in this paper, neutralization of IL-17 markedly reduces expression of certain proinflammatory molecules such as IL-6, IL-8, and CCL20 within the graft while leaving expression of CXCL10, an IFN-γ–inducible chemokine, unaffected. IL-6, CXCL8, and CCL20 are known IL-17–inducible genes (27
); however, the efficacy of IL-17 neutralization is somewhat surprising given that IL-17 is far less potent at inducing expression of these molecules than IL-1 or TNF (unpublished data). These data indicate that in the absence of other human leukocytes, allogeneic T cell–mediated induction of these acutely inflammatory transcripts requires the action of IL-17. Our finding that IL-17 synergizes with TNF to induce CCL20 production by cultured SMCs suggests that these factors may act in concert within the vessel wall to drive chemokine expression and that interference with one single factor can strongly reduce CCL20 production.
The reduction in CCL20 expression caused by IL-17 neutralization appears functional because it is associated with a corresponding decrease in CCR6 expression within the graft, suggesting that alloreactive T cell–derived IL-17 propagates an inflammatory cascade that promotes further accumulation of inflammatory, CCR6+
T cells during human allograft rejection. Because our conclusion is based on mRNA levels, we cannot distinguish whether this reduction in CCR6+
cells represents a change in the number of CCR4+
T cells that produce only IL-17 or the number of cells in the CCR6+
population that produce both IL-17 and IFN-γ (25
). Interestingly, IL-1 blockade did not cause a similar specific reduction in CCR6 expression in rejecting artery grafts, which may suggest that a more complete inhibition of IL-17, as achieved with antibody neutralization of IL-17 compared with treatment with IL-1Ra, is required to affect subsequent CCR6+
T cell recruitment. IL-17–induced production of chemokines that recruit neutrophils (CXCL8) and DCs (CCL20) observed in the experiments presented in this paper would be expected to contribute to the rejection of human allografts; however, more complete human–mouse chimeric models that allow for the engraftment or development of human neutrophils and DCs will be required to further evaluate these effects.
The effects of neutralizing IL-17 on overall graft pathology are less than those observed by blocking IL-1 and much less than those observed by blocking IFN-γ (52
). Indeed, IFN-γ still appears to be the central and nonredundant effector cytokine in our model. Nevertheless, our results suggest that IL-1 blockade may be a useful adjunct therapy to prevent vascular rejection, an idea supported by results in mouse and rat cardiac transplant models showing delayed acute rejection with IL-1 blockade (53
). As IL-1 is well known to be produced as a consequence of ischemia-reperfusion injury (55
), we propose that IL-1 produced in the periphery as a consequence of perioperative injury promotes T cell–mediated inflammation locally within an allograft and further modulates the nature of the T cell response. Perioperative blockade of IL-1 thus represents an attractive new therapeutic strategy in clinical transplantation that will need to be tested experimentally. This role of IL-1 on T cell responses may also carry significant implications for autoimmunity, tumor immunotherapy, and stroke.