Allogeneic haematopoietic cell transplantation (HCT) is a transplant between two genetically non-identical individuals (Ferrara et al., 2009). It is an important therapeutic option to treat various malignant and non-malignant diseases, including acute and chronic leukemias, myelomas, lymphomas, aplastic anemias, solid tumors, and severe immunodeficiency disorders (Goker et al., 2001). The number of allogeneic HCTs performed annually worldwide is estimated to be more than 20,000 (Choi et al., 2010). After allogeneic HCT, some patients present with remission of primary disease. In some cases, patients may develop a common secondary complication in which transplanted cells reject the host tissues, named graft versus host disease (GVHD). GVHD accounts for 15–30% of deaths following allogeneic HCT and is a major cause of morbidity in up to 50% of transplant recipients (Ferrara et al., 2009).

Graft versus host disease manifests in two different forms, acute (aGVHD) and chronic (cGVHD). Acute GVHD occurs within 100 days of allogeneic HCT and is a rapidly progressive syndrome that is characterized by profound wasting, immunosuppression, and tissue injury in a number of organs, including the intestine, spleen, skin, liver, and lung (Howard and Woodruff, 1961; Lapp et al., 1985; Cooke et al., 1998; Wysocki et al., 2005; Socié and Blazar, 2009). In aGVHD, cytokines stimulate donor T cells to recognize host antigens that are presented by antigen-presenting cells (APCs). These T cells become activated and migrate to target organs where they generate effector responses against the host (reviewed by Wysocki et al., 2005; Jaksch and Mattsson, 2005). Unlike aGVHD, cGVHD occurs usually 100 days after bone marrow transplantation (BMT) and resembles an autoimmune syndrome (Will and Wynn, 2006). In addition to the effects mediated by T cells, cGVHD involves B-cell stimulation, autoantibody production, and systemic fibrosis (Schroeder and DiPersio, 2011). Although donor T cells may mount an effector response against the host cells, these cells also play a very important role in preventing the recurrence of the original malignant disease, especially when the HCT is given as a therapy for leukemia. These types of responses are referred to as graft versus leukemia (GVL; Johnson et al., 1996, 1999; Kolb, 2008). Thus, the inhibition of GVHD without interfering with GVL is of major interest therapeutically.

The management of GVHD is an old problem but is still unresolved. Standard therapy for GVHD includes high doses of corticosteroids, but the success of this therapy is not great, as mortality rates are more than 40% (Devetten and Vose, 2004; Ferrara and Reddy, 2006; Ferrara et al., 2009; Choi et al., 2010). In addition, patients that develop corticosteroid refractory GVHD have a high risk of death due either to GVHD itself or to secondary infections (Ferrara and Reddy, 2006). Although new therapies, including monoclonal antibodies against the IL-2 receptor (daclizumab), the TNF-α receptor (entanercept), or TNF-α (infliximab), and immunosuppressive drugs, such as mycophenolate mofetil, have been proposed to treat GVHD, these therapies are still not satisfactory (Choi et al., 2010). A better understanding of the mechanisms involved in the pathogenesis of GVHD may yield novel therapeutic targets. The present review discusses the role of chemokines and their receptors during GVHD.

Chemokines are a family of small proteins (about 8–14 kDa) that are classified into four major groups based on the number and spacing of conserved cysteines; the groups include the CC group (CCL1–28), the CXC group (CXCL1–16), the C group (XCL1–2), and the CX3C group (CX3CL1; Murphy et al., 2000; Murphy, 2002). Chemokines exert their effects through interaction with one or more members of a family of seven transmembrane domain-containing G-protein-coupled receptors (Zlotnik and Yoshie, 2000; Rot and Von Andrian, 2004; Charo and Ransohoff, 2006). There are currently 10 identified CC chemokine receptors (CCR1–10), 6 CXC receptors (CXCR1–6), 1 C receptor (XCR1), and 1 CX₃C receptor (CX₃CR1; Murphy et al., 2000; Murphy, 2002; Kittan and Hildebrandt, 2010; Russo et al., 2010). Chemokine expression can be enhanced by inflammatory cytokines (Mackay, 2001), and chemokines have an important role in recruiting cells of the innate and adaptive immune system to sites of inflammation (Rollins, 1997; Moser et al., 2004). In addition, chemokines have been suggested to be important for leukocyte activation (Choi et al., 2007), angiogenesis (Addison et al., 2000; Strieter et al., 2005), haematopoiesis (Broxmeyer, 2008), and the organization and function of secondary lymphoid tissues (Cyster, 1999; Muller et al., 2003; Ohl et al., 2003).

Understanding of the molecular mechanism involved in controlling expression of chemokine and their receptors in GVHD may provide efficient strategies to manage of disease. However, little is known about such mechanisms. Most studies report that the conditioning regime are a initial signal to trigger production of cytokines (such as TNF-α, IFN-γ, IL-1, IL-2) and, consequently, up regulation of chemokine receptors and their ligands (Krenger et al., 1997; Jaksch and Mattsson, 2005; Wysocki et al., 2005; Mapara et al., 2006; Bouazzaoui et al., 2009; Kittan and Hildebrandt, 2010). TNF-α and IFN-γ are produced during the initial phase of GVHD within lymphoid tissues and may induce production of chemokines in target organs by host cells (Schroeder and DiPersio, 2011). IFN-γ is crucial for differentiation of CD4⁺ T cell into Th1 cells which increase the expression of CCR9, CCR5, and CXCR6u and their ligands in intestine and liver (Yi et al., 2009). IL-2 is another important cytokine involved in T cell activation and expansion (Jaksch and Mattsson, 2005) and influences production of pro-inflammatory chemokines such as CCL2, CCL3, CCL4, CCL5 (Jaksch and Mattsson, 2005; Wysocki et al., 2005; Choi et al., 2007; Castor et al., 2010; Kittan and Hildebrandt, 2010). Therefore, the conditional regime and the cytokines associated with activation of T cells will provide the necessary stimuli for the production of chemokines, which in turn will promote and orchestrate the recruitment of immune cells during all phases of GVHD. Here, we reviewed chemokines involved in the pathogenesis of GVHD and discuss recent studies that have shown that interference in the chemokine system using antibodies and compounds may decrease the severity of GVHD while preserving the GVL response.

The pathogenesis of acute GVHD is currently understood as a three-phase response. The first phase is associated with the conditioning regimen (irradiation/chemotherapy) that leads to damage of host tissues, including the intestinal mucosa and liver (Ferrara, 1993; Hill et al., 1997; Jaksch and Mattsson, 2005; Mapara et al., 2006). The second phase is characterized by activation and proliferation of donor T cells. After transplantation, donor T cells interact with host APCs, recognize host antigens, become activated, and differentiate into effector cells (Jaksch and Mattsson, 2005; Wysocki et al., 2005). The greater the disparity between donor and recipient major histocompatibility complex (MHC), the greater the T cell response will be (Socié and Blazar, 2009). The interaction of T cells with APCs usually occurs in secondary lymphoid organs, including the spleen and lymph nodes (Zhang et al., 2005), but it can also occur in other peripheral lymphoid tissues, such as Peyer’s patches (PP; Murai et al., 2003). In the third phase of the acute GVHD response, activated T cells migrate to target organs (intestine, liver, lung, and skin) and release cytolytic molecules and inflammatory cytokines, such as IFN-γ and TNF-α, and undergo Fas/Fas ligand interactions (Baker et al., 1996; Braun et al., 1996; Cooke et al., 1998; Schmaltz et al., 2001; Jaksch and Mattsson, 2005). Recruitment of other effector leukocytes, including macrophages, follows T cell migration, and this process is thought to be important for the perpetuation of inflammatory responses and the destruction of target organs (Socié and Blazar, 2009; Castor et al., 2011; Schroeder and DiPersio, 2011).

Although the migration of T cells into secondary lymphoid organs during GVHD has been well characterized (Wysocki et al., 2005), the migration of leukocytes into parenchymal organs is less well understood. The latter process depends on interactions between selectins (Ley and Kansas, 2004; Luster et al., 2005; Carlson et al., 2010; Lu et al., 2010) and integrins (Hogg et al., 2002, 2003) and their ligands as well as on chemokine–chemokine receptor interactions (Wysocki et al., 2005; Castor et al., 2010; Kittan and Hildebrandt, 2010). Animal models of GVHD have provided important insights into the three characteristic phases of aGVHD.

Several studies have now described there is increased expression of chemokines and chemokine receptors in GVHD (New et al., 2002; Jaksch and Mattsson, 2005; Mapara et al., 2006; Bouazzaoui et al., 2009; Castor et al., 2011). The profile of chemokine and chemokine receptor expression is different in different target organs of GVHD. Table 2 and Figure 1 summarize the expression of chemokines and chemokine receptors in GVHD in various target organs and during different temporal phases of the disease.

Signaling by chemokine receptors is mediated by heterotrimeric G-proteins (Horuk and Proudfoot, 2009). Activation of G-proteins leads to activation of protein and lipid kinases, including mitogen-activated protein (MAP), Janus kinase-signal transducer and activator of transcription (JAK-STAT), and phosphatidyl inositol-3-kinase (PI₃K), which mediate actin cytoskeleton rearrangement, changes in integrin affinity and avidity, leukocyte migration and proliferation, and cellular differentiation and apoptosis (Ribas et al., 2007; Russo et al., 2010).

Recent studies have attempted to elucidate the role of molecules downstream of chemokine receptor signaling and to establish a functional hierarchy involved in the development of GVHD, represented in Figure 2 (Cetkovic-Cvrlje et al., 2001, 2002; Cetkovic-Cvrlje and Uckun, 2004; Sun et al., 2004, 2005; Hill et al., 2010; Park et al., 2010; Castor et al., 2011; Ma et al., 2011). Modulation of these downstream signaling molecules is an alternative way to interfere with the chemokine/chemokine receptor system.

We have recently evaluated the role of PI₃Kγ in the development of GVHD (Castor et al., 2011). PI₃Kγ in donor cells was relevant for the initial surge of chemokine production in the target organs of mice subjected to GVHD. In addition to production of pro-inflammatory mediators in target tissues, infiltration of CD4⁺, CD8⁺, and CD11c⁺ cells was decreased with the absence of PI₃Kγ in donor cells, and pharmacological blockade of PI₃Kγ was associated with decreased rolling and adhesion of leukocytes to target organs as assessed by intravital microscopy. These effects on cell recruitment were translated as overall clinical improvement and decreased lethality in the absence of PI₃Kγ or its pharmacological inhibition in donor cells (Castor et al., 2011).

Phosphorylation of ERK-1/2 and STAT-3 are involved in important events during T cell (allo) activation in GVHD, and interference with STAT-3 phosphorylation can inhibit T cell activation and proliferation in GVHD both in vitro and in vivo (Lu et al., 2008). Additionally, expansion of CD4⁺ and CD8⁺ T cells depends on the expression of phospho[p]-STAT-1 and p-STAT-3. GVHD-specific STAT-3/STAT-1 activation preceded the activation of nuclear factor-κB (NF-κB) and MAP kinases and was associated with the subsequent expression of interferon regulatory factor-1 (IRF-1), suppressor of cytokine signaling 1 (SOCS-1) and IL-17 (Ma et al., 2011). STAT-1 expression in the spleen preceded its expression in target organs and was correlated with the chemokine storm in these organs. STAT-3 expression was similar to that of STAT-1 and was observed early in secondary lymphoid organs and later in target tissues. In the spleen, STAT-3 expression was correlated with high levels of IL-6 and IL-10. The marked change in the IL-6/IL-10 ratio during the development of GVHD suggests that STAT-3 may act as a promoter of inflammation during the early priming and induction phase of GVHD (high IL-6/IL-10 ratio) but may mediate anti-inflammatory signals at later time points (low IL-6/IL-10 ratio; Ma et al., 2011). By contrast, early inhibition of NF-κB may reduce GVHD by affecting primarily the haematopoietic compartment with inhibition of donor T cell expansion or host APC maturation. However, delayed inhibition of NF-κB may interfere with target tissue regeneration or promotion of inflammation, leading to worsening of GVHD (Sun et al., 2004, 2005). Interestingly, cytokine signaling through JAK-STAT-3 in GVHD was regulated by SOCS-3 (Hill et al., 2010). Transplantation of donor T cells into SOCS-3 deficient mice led to persistent phosphorylation of STAT-3, resulting in enhanced T cell proliferation, greater Th1 and Th17 differentiation, and production of IFN-γ and IL-17 (Hill et al., 2010). Thus, SOCS-3 has a regulatory effect and is an attractive target for GVHD therapeutic modulation; functional augmentation of SOCS-3 may preferentially inhibit alloreactive T cell proliferation and differentiate cells away from pathogenic Th17/Th1 pathways. Janus kinase signaling occurs downstream of chemokine receptor signaling, and there are molecules that inhibit this pathway. One such inhibitor, CP-690550, was found to decrease mortality and reduce target organ damage in mice subjected to GVHD by suppressing donor CD4⁺ T cell-mediated (IFN)-γ production and inhibition of Th1 differentiation (Park et al., 2010). Specific inhibitors of Janus kinase 3 (JAK-3) have already been tested as a treatment for GVHD. The use of the JAK-3 inhibitor, WHI-P131 [4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline], showed improved mortality rates and decreased liver and skin damage (Cetkovic-Cvrlje et al., 2001; Uckun et al., 2002). Another JAK-3 inhibitor, 4-(3′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (JANEX-3), improved mortality rates and ameliorated the clinical symptoms of GVHD (Cetkovic-Cvrlje et al., 2002). A specific Bruton’s tyrosine kinase (BTK) inhibitor (LFM-A13), was also tested as a treatment for GVHD; treated mice showed increased survival rates and had less clinical GVHD. The combined treatment of LFM-A13 with JANEX-3 was more effective than treatment with LFM-A13 or JANEX-3 alone (Cetkovic-Cvrlje and Uckun, 2004). Taken together, these results indicate that signaling molecules downstream of chemokine signaling may be useful targets for treating GVHD.

In the context of the treatment of hematological malignances, such as leukemia, engraftment of donor cells is important to restore the immune system after ablative therapy (Kolb, 2008). In addition to reconstructing the immune system, the engrafted cells are thought to contribute to chemotherapy by inducing an anti-tumor effect, an effect that is known as (GVL; Rezvani and Barrett, 2008). Several therapies that decrease GVHD may decrease GVL, which is an undesirable outcome of such therapies (Rezvani and Barrett, 2008). Therefore, it is generally accepted that, in the context of haematopoietic stem cell transplantation, a therapy should decrease or prevent GVHD but ideally should not modify the associated GVL (Kolb, 2008; Rezvani and Barrett, 2008).

Although the chemokine system represents a promising system to target to develop new GVHD therapies, it is also important to understand the role of chemokines in GVL response. Evaluation of GVL has not been the major focus of studies involving chemokines and GVHD. However, we have found a few studies showing that, by interfering with the chemokine system, it is possible to decrease GVHD without interfering with GVL.

Our group (Castor et al., 2010) and Choi et al. (2007) demonstrated that, despite the important action of CCR1 and its ligands, CCL3, and CCL5, in the GVHD response, neutralization of CCL3, or the absence of CCR1 in donor cells did not interfere with GVL (Castor et al., 2010; Choi et al., 2007). The capacity of T cells to eliminate tumor cells remained unaltered upon neutralization of CCL3 by evasin-1 in mice subjected to GVHD (Castor et al., 2010). The absence of CCR1 in donor cells also maintained the GVL response in mice subjected to GVHD (Choi et al., 2007). Ueha et al. (2007) verified the GVL response in a study investigating the role of fractalkine (CX3CL1) in GVHD. In this study, CX3CL1 was important for GVHD development, but not for the GVL response, and treatment with anti-CX3CL1 decreased GVHD without modifying GVL. The same result was observed when a downstream chemokine receptor molecule, PI₃Kγ, was absent in donor cells. Transplantation of PI₃Kγ-deficient splenocytes reduced the ability of these cells to react against the host, but not against the tumor (Castor et al., 2011).

The results described above indicate that the clinical use of inhibitors of these molecules may decrease the GVHD reaction but not interfere with GVL responses.

The explicit participation of chemokines in the pathophysiology of different diseases has initiated the development of pharmacological strategies that can interfere with the chemokine system. Chemokines function by signaling through seven transmembrane G-protein-coupled receptors, which are one of the most druggable classes of receptors in the pharmaceutical industry (Horuk and Proudfoot, 2009).

Since 1996, interest in targeting the chemokine system has been growing, especially after demonstration of the participation of CCR5 as a co-receptor of HIV infection (Liu et al., 1996; Samson et al., 1996). After those studies, the pharmaceutical industry began investing in the development of molecules that could interfere with chemokine/chemokine receptor interaction. Examples of such molecules include chemokine receptor antagonists (Hesselgesser et al., 1998; White et al., 1998; Horuk and Proudfoot, 2009; Garin and Proudfoot, 2011), modified chemokines that act as antagonist molecules (Proudfoot et al., 1999, 2010), neutralizing antibodies to the chemokines or their receptors (Gonzalo et al., 1998) and chemokine binding proteins (Alcami, 2003; Smith et al., 2005; Frauenschuh et al., 2007; Déruaz et al., 2008; Russo et al., 2010). In 2007, the FDA approved maraviroc, an inhibitor of CCR5 for the prevention of HIV infection, which was the first triumph for a small-molecule drug acting on the chemokine system. A second small-molecule drug, a CXCR4 antagonist for haematopoietic stem cell mobilization, was approved by the FDA at the end of 2008. The results of a Phase III trial with a CCR9 inhibitor for Crohn’s disease are also promising. The latter drug could represent the first success for a chemokine receptor antagonist to be used as an anti-inflammatory therapeutic. Development of this small-molecule drug confirms the importance of chemokine receptors as a target class for anti-inflammatory and autoimmune diseases (Proudfoot et al., 2010; Garin and Proudfoot, 2011).

There are many difficulties in translating beneficial results from murine studies to humans, one of which is the many caveats and differences between disease in experimental models and humans. Humans undergoing BMT have a primary disease and are subjected to immunosuppressive treatments before and during the transplantation. The usual conditioning regimen in humans, which consists of chemotherapy and radiation, is not always used. The source of donor cells and genetic and immunological disparities are also different from most animal models. Infectious challenges are not usually performed in conjunction with experimental induction of GVHD, but infections are commonly observed in immunosuppressed patients. Human microbiota is markedly different from the microbiota of a mouse kept in a pathogen-free facility, and bacterial translocation and sepsis are important causes of death in GVHD patients. Finally, young mice are usually used in experimental GVHD induction, but GVHD is generally more common in older people. These differences should not hamper development of drugs against GVHD but do not need to be taken into consideration when moving drugs forward into clinical trials.

Fewer studies have been performed to validate the use of inhibitors of the chemokine system in experimental GVHD. In this context, Evasin-1 (CCL3-CBP), CXCR3 antagonists, anti-CX₃CL1, inhibitor of CCR5 and CCR9 (CCX282), oligopeptides, such as NR58-3143, and inhibitors of molecules involved in downstream signaling of chemokine receptors (PI₃Kγ, STAT-1 and 3, JAKs, and SOCS-1 and 3) decrease GVHD in mice and may hence represent an interesting clinical approach in humans. However, to the best of our knowledge, there are no studies confirming the effects of inhibitors of the chemokine system in GVHD in humans. Many experimental studies have not clarified the mechanism by which abrogation of inflammatory responses occur after use of therapies based on chemokine inhibition. Therefore, more mechanistic studies are needed to understand in greater detail the use of these therapeutic molecules in experimental GVHD. As mentioned above, any therapy for GVHD should decreased clinical disease but not interfere with GVL. In this respect, strategies based on CCL3, CCL5, and CX₃CL1 appear to be the most promising approach based on the existing experimental systems.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.