The role of complement in the trafficking of hematopoietic stem/progenitor cells

Transplantation of hematopoietic stem/progenitor cells (HSPCs) has become a well-established treatment for various malignant and nonmalignant hematologic disorders and certain solid tumors. Approximately 60,000 autologous and allogeneic HSPC transplants are performed annually worldwide. Characteristics of HSPCs that make clinical transplantation feasible, apart from their regenerative potential, include their ability to be coaxed out of the bone marrow (BM), or “mobilize,” and their capacity to “home” back to the marrow space after intravenous (IV) infusion. In our investigations of HSPC mobilization and homing, the complement system has emerged as an important, yet underappreciated, modulator of this bidirectional trafficking of HSPCs. Although many factors contribute to HSPC mobilization and homing, here, we focus on the role of complement cascade (CC) components C1q, C3a, C5a, and C5b-C9 (membrane attack complex [MAC]) and infer that their modulation in the future could have significance to improve outcomes of HSPC transplantation. After autologous and allogeneic transplantation the patient requirements for red blood cell transfusion are high (median, 12 units; range, 8-16 units per patient),1 and despite variations in practice, there is also substantial need for platelet (PLT) transfusions (median, 5 units; range, 0-110 units per patient).2 Hence, improving the strategies for HSPC collection based on better understanding of the mechanisms of mobilization and homing could also reduce the utilization of blood products. Because HSPCs reside primarily in the BM, HSPCs for both autologous and allogeneic transplantation were traditionally collected by means of multiple aspirations from the posterior iliac crest under general anesthesia. BM transplantation was pioneered in the 1950s by a team led by E. Donnall Thomas, who showed that BM-derived stem cells infused IV repopulate the recipient BM and reconstitute hematopoiesis.3 In the late 1970s it was shown that during steady-state homeostasis, a small number of HSPCs circulate continuously in the human peripheral blood (PB) and this number increases after treatment with chemotherapy (e.g., with cyclophosphamide) and/or growth factors and cytokines (e.g., granulocyte–colony-stimulating factor [G-CSF]) that mobilize HSPCs from BM into the PB.4,5 Currently, mobilized (m)PB HSPCs have almost completely replaced HSPCs from BM for autologous and three-quarters of allogeneic transplantations.6,7 Collection of mPB HSPCs by leukapheresis is carried out in an outpatient setting and is therefore less invasive and without the risks associated with general anesthesia. Moreover, randomized trials have shown that neutrophil and PLT engraftment generally occurs faster after mPB transplantation than after BM transplantation, likely due to the higher number of HSPCs collected in mPB and transplanted.8 Another possible explanation is that HSPCs from mPB are exposed to CC cleavage fragments (e.g., C3a) during leukapheresis and collection and to cationic bioactive peptides released from granulocytes (e.g., LL-37).9-12 More rapid engraftment reduces risk of infection, number of transfusions, and length of hospitalization. However, donor-patient responses to mobilizing agents vary; up to 5% of healthy allogeneic donors mobilize poorly and up to 60% of high-risk patients failed to mobilize at all, depending on their underlying disease, prior chemotherapy regimens, age, and other factors.7 An alternative to BM or mPB as source of HSPCs is umbilical cord blood (CB). Since the first CB transplant in 1988, an estimated more than 30,000 CB transplants have been performed worldwide in both pediatric and adult patients.13-15 However, the main limitation of CB transplantation use in adults is the low HSPC (CD34+ cell) dose available in one CB unit, which is generally insufficient to support engraftment in adult patients. Retrospective analysis of CB transplantation outcomes in adults has shown delayed neutrophil engraftment (27 days with CB vs. 18 with BM) and PLT engraftment (60 days with CB vs. 29 with BM).15 Currently, efforts are being made to elucidate the mechanisms of HSPC homing and develop new strategies promoting more efficient hematopoietic reconstitution. These include use of more than 1 CB unit for transplantation, ex vivo expansion, and intrabone infusion.16-18 In this review we focus on the complement system as a means for enhancing homing of CB HSPCs. Current perception of the processes of HSPC mobilization and homing derives from our understanding of the dynamic interactions between HSPCs and the BM microenvironment, which comprise the stem cell niche. The concept of niches as first proposed by Schofield19 describes three-dimensional spatially organized anatomical compartments in the BM where stem cells reside and are maintained. Mounting evidence later revealed that the BM niche provides not only a simple static structural support but also topographic information and the appropriate physiologic cues to control the dynamic balance of stem cell quiescence, self-renewal, differentiation, and apoptosis, as well as HSPC localization and migration.20,21 The existence of the endosteal/osteoblastic and the vascular niches has been suggested. The endosteal/osteoblastic niche close to the bone, a site of relative hypoxia where immature osteoblasts are in close contact with HSPCs, plays a major role in the maintenance of hematopoietic stem cell quiescence.22-24 The vascular niche consisting of sinusoidal vessels provides a microenvironment rich in nutrients, growth factors, and oxygen and plays a role in hematopoietic stem cell proliferation and differentiation and ultimately the egress of mature progenitors into the circulation.22,23,25 HSPC mobilization is primarily mediated by alterations in the cellular components of the BM niche.26 Perivascular mesenchymal stem cells (MSCs), macrophages, sinusoidal endothelial cells, osteoblasts, and sympathetic nerve fibers form the niches that harbor HSPC during homeostasis and mediate their egress in response to mobilizing agents.21,27,28 For example, suppression of resident monocytes-macrophages leads to decreased expression of factors required for HSPC retention and results in HSPC mobilization.29 Furthermore, depletion of endosteal macrophages (osteomacs) that form a canopy over mature osteoblasts at sites of bone formation and support osteoblast function elicited robust mobilization of HSPCs, suggesting that BM macrophages play a critical role in the maintenance of endosteal HSPC niches.30 On the other hand, it was recently demonstrated that nestin-expressing perivascular MSCs are either in direct contact with HSPC or in clusters around them. These nestin+ MSCs express HSPC maintenance genes and, upon their deletion, significant reduction in BM HSPC is observed, owing at least in part to their mobilization toward extramedullary sites.31 The BM is highly innervated, with nerve fibers running along blood vessels, and increasing evidence indicates a major role for signals coming from the sympathetic nervous system in the regulation of HSPC retention, homing, and mobilization.27,31 The cellular components of the BM microenvironment transmit and receive signals through soluble factors (e.g., growth factors, cytokines and chemokines, hormones), bioactive lipids, cell adhesion molecules, extracellular matrix (ECM), neural inputs, and the vascular network. Stromal cell–derived factor (SDF)-1 (also known as CXCL12) is a chemokine that strongly attracts HSPCs, which express its receptor CXCR4.32,33 SDF-1 is constitutively expressed at high levels by osteoblasts and endothelial cells and represents a potent retention signal for HSPCs. HSPCs are also retained in the niche by adhesion molecules acting through cell-to-cell contact (e.g., via the very late antigen [VLA]-4/vascular cell adhesion molecule [VCAM]-1 axis) and attachment to ECM components. With the HSPC firmly entrenched in the BM niches, it can be envisioned that their mobilization to PB would require the breaking down of adhesive interactions, alterations in chemotactic gradients (e.g., an increase in the sphingosine-1 phosphate [S1P] gradient in BM sinusoids) and proteolysis of ECM and other molecules that promote anchorage of HSPC to their niches (e.g., those released from granulocytes and monocytes stimulated by G-CSF or the C5a CC cleavage fragment).10,34 During steady-state hematopoiesis, the continuous traffic of HSPCs between the BM and PB, albeit at a very slow rate, contributes to maintaining normal hematopoiesis. However, stress conditions such as inflammation or injury greatly amplify the egress of HSPCs from the BM. These processes are mimicked in clinical mobilization in which HSPCs are recruited from the BM to PB by means of pharmacologic agents and collected for use in transplantation.5-7 Several cytokines, growth factors, and chemokines (G-CSF, granulocyte macrophage [GM]-CSF, Flt-3 ligand, interleukin [IL]-8, stem cell factor [SCF], hepatocyte growth factor [HGF], SDF-1, and GROβ) can trigger mobilization in varying degrees.35-37 Recent studies have demonstrated that thrombolytic agents, such as microplasmin, tenecteplase, and recombinant tissue plasminogen activator, enhance G-CSF–induced mobilization in murine models,38 and the proteolytic enzyme membrane type 1-matrix metalloproteinase (MT1-MMP) is up regulated by G-CSF and contributes to ECM degradation, thus enhancing the migration of HSPCs.39,40 HSPCs can also be easily mobilized by CXCR4 receptor antagonists (AMD3100 also known as plerixafor, T140) or agonists (CTCE-0021, ATI-2341) and after blockage of VLA-4 integrin on HSPC by BIO 4860.10,11,41,42 Conversely, homing occurs when transplanted HSPCs travel from the blood circulation and establish residence within the BM niche. Early on, the homing process was described as the “rolling, crawling, and nesting” of HSPCs into the marrow stromal space.43 Even then, seminal studies had already identified the significant contributions of a wide variety of adhesion molecules and their receptors in mediating cell-to-cell and cell-to-matrix interactions, and the role of the SDF-1/CXCR4 axis in the retention of HSPC in the BM niche was also realized.44-47 It soon became evident that proteolytic enzymes capable of degrading ECM components could play a role in the migration of HSPCs.48 It is now recognized that homing is a complex multistep process that involves signaling through adhesion molecules; chemotactic molecules; and their receptors, proteases, and other factors. In addition, GM-CSF, IL-3, and SCF, which activate VLA-4 and VLA-5 and increase the adhesiveness of HSPC, as well as Flt3-ligand, SCF, IL-3, IL-6, and HGF, which in addition to prostaglandin E2 (PGE2), up regulate the expression of CXCR4 on HSPC, also affect the homing of HSPC.33 A flexible hierarchy of cooperating homing pathways with the dominant players characterized by significant functional overlap and constant repositioning within changing cytokine milieus has been postulated.49 Thus, existing empirical evidence demonstrates the interplay of cellular components and various signaling molecules in modulating HSPC trafficking. Our recent research has shown that activation of the CC takes place in both the mobilization and homing of HSPCs, and we discuss it below in detail. The human CC has been traditionally recognized as a supportive first line of host defense against infections. It is now known that its functions extend far beyond the elimination of foreign bodies by acting as a rapid and efficient immune surveillance system that discriminates between healthy host tissue, cellular debris, apoptotic cells, and pathogenic microbes and responds accordingly.50,51 The complement proteins are important elements of the innate immune response that act in a cascade to induce their physiologic effects. Three main pathways for complement activation are recognized, namely, the classical, alternative, and lectin pathways, as recently reviewed.50,51 The classical pathway, often referred to as the antibody (IgG)-dependent pathway, is initiated by noncovalent binding of the three-subunit component 1, composed of C1q, the recognition subunit of the complex, and two chains each of C1r and C1s, proteases that are activated upon surface binding of C1q. Activated C1 cleaves C4 and C2 releasing smaller fragments (C4a and C2b) and larger fragments (C4b and C2a). C2a coordinates with C4b to form an enzymatic complex termed C3 convertase, with the ability to cleave C3 into larger C3b and smaller C3a, an anaphylatoxin with proinflammatory properties (Fig. 1).50 Deposition of C3b on the target surface induces the formation of C5a convertase, which cleaves C5 into C5b and C5a, another anaphylatoxin. C6, C7, C8, and C9 bind serially to C5b to form the MAC, which initiates cell lysis when inserted into the cell membrane of an invading cell. The alternative (IgG-independent) pathway is triggered by spontaneous C3 hydrolysis, which exposes its internal thioester group to form C3a and C3b. Upon covalent binding to a pathogenic membrane C3b is bound by Factor B to form a complex, which, in the presence of Factor D, is cleaved into Ba and Bb. Bb remains covalently bonded to C3b to form C3bBb, which has proteolytic activity, catalyzing the hydrolysis of C3 in the blood into C3a and C3b. Deposition of C3b molecules leads to the formation of a C5 convertase, which cleaves C5 into C5a and C5b, as in the classical pathway (Fig. 1).50 The lectin pathway is homologous to the classical pathway, but with the opsonin, mannose-binding lectin (MBL), and ficolins, instead of C1q. This pathway is activated by binding MBL to mannose residues on the pathogen surface, which activates the MBL-associated serine proteases MASP-1 and MASP-2 (very similar to C1r and C1s, respectively), which can then split C4 into C4a and C4b and C2 into C2a and C2b. C4b and C2b then bind together to form the C3-convertase, as in the classical pathway.50 Because all three complement pathways merge with C3b deposition on a target and C3b is the initiating factor of the alternative pathway, complement activation can be initiated by the classical or lectin pathway and amplified by the alternative pathway. CC activation during HSPC mobilization. The CC may become activated through the classical pathway (which is dependent on naturally-occurring antibodies [NA-Ig] and is triggered by complement protein C1q), the lectin pathway, and the alternative pathway (which is triggered by Factors B and D). All three pathways merge at complement protein C3, whose activation leads to the release of C3 anaphylatoxin and complement protein C5. C5 is enzymatically cleaved to release anaphylatoxin C5a. Some alternative mechanisms exist, whereby C5 could be activated by the proteases thrombin and kallikrein. The arginine terminal residue of both C3a and C5a is cleaved to and which have C3a and cleavage fragments enhance the of HSPC to the BM to a factor (SDF)-1 thus promoting their retention in the BM, C5a and the egress of granulocytes and the for the mobilization of HSPCs. The of CC activation is the of the C5b-C9 The C3a and C5a are released and trigger signals through their receptors, and (also may also an alternative is expressed on and and the of and is expressed on and as well as and endothelial initiated in granulocytes and monocytes via include of and up adhesion molecules, and of oxygen of granulocytes and monocytes by C5a and its play an important role in the release of proteolytic enzymes that of HSPCs from their Furthermore, three receptors for have been which a and which the of The triggered by include the of and in cells and expression of adhesion molecules in endothelial cells and of It is that the complement receptors are in the signaling pathways by the complement component Because HSPCs could be recruited to the peripheral during or injury as an important part of innate we HSPC mobilization is regulated by elements of innate in by CC on the of HSPC mobilization have been together with investigations of strategies to improve HSPC on a review of clinical trials cytokines, or for mobilization of HSPCs, it was that the mobilization is interactions between HSPCs and the BM have been identified and have become to enhance mobilization. It is important to that mobilization could have on the (e.g., expression of genes in cell apoptosis, cell and which may affect the of HSPC The complement system is an component of innate that we recently to be one of the major players in HSPC it has been that G-CSF–induced mobilization is in patients from that who functional B and we proposed that the egress of HSPCs from the BM occurs as part of the immune Since the classical pathway of CC is activated by a of as mobilization in patients could be by of that the antigen on BM cells expressed during mobilization. are also due to their of the of expression of on BM cells during and activation of it was that mobilizing agents (e.g., granulocytes and monocytes in BM to release proteolytic enzymes (Fig. that the BM microenvironment into a proteolytic This can be as a of to of on BM an activation of the The antibodies that circulate in the PB and, via C1q, CC activation through the classical pathway C3 and C5 cleavage fragments (Fig. This of CC activation is for G-CSF–induced mobilization. of of the interactions in response to G-CSF–induced mobilization. HSPCs are retained in the BM niche by adhesive interactions, such as between and and chemotactic interactions, such as between SDF-1 and CXCR4 signals (e.g., the number of granulocytes and activate the C5 cleavage fragments activate cells via the C5a receptor to release proteases which and SDF-1/CXCR4 on the surface of granulocytes and HSPCs and their In addition, C5 cleavage fragments granulocytes and the for the mobilization of HSPC the in the of CC activation the release of from is also a potent for HSPCs, which express the In (e.g., activate the CC by the alternative pathway, which in various that C3 and C5 cleavage fragments (Fig. C3 could also be cleaved by proteases released from granulocytes after of mobilizing In the of C3 cleavage fragments in the BM increases during In addition, the proteolytic of thrombin and also activate C5 (Fig. shown in 1, CC cleavage fragments play in the mobilization of HSPCs. C3 cleavage fragments mobilization by promoting BM retention, C5 cleavage fragments and the activation of C5b-C9 promote mobilization. This is by the that C3 cleavage are C5 cleavage fragments and not mobilize very demonstrated that C3 cleavage fragments enhance and/or the of HSPCs to an SDF-1 thus promoting retention of cells in the In as studies as well as studies C3a revealed that the axis HSPCs against their egress from the BM and that the of this axis with the increases HSPC On the other hand, poorly to G-CSF–induced mobilization to their suggesting an important role of the part of the CC in promoting egress of HSPCs from the BM into elucidate the role of C5 cleavage fragments in the mobilization of human HSPCs, we the levels of the cleavage that levels are higher and with cell and blood cell in patients who are Although C5 cleavage fragments not progenitors strongly both granulocytes and monocytes (Fig. the C5a receptor was not on cells but on more mature and Moreover, we that and cells from PB had a higher of cells than those from their PB Furthermore, C5a of granulocytes and monocytes decreased CXCR4 expression and toward an SDF-1 gradient and of expression of and by cells (Fig. These support the that C5 cleavage fragments the egress of HSPCs into mPB by increasing the of proteolytic enzymes from the function of the SDF-1/CXCR4 and (Fig. C5 cleavage fragments also granulocytes that the for egress of HSPC (Fig. of the part of the CC is also in C5b-C9 the activation of which leads to the release of into BM from red blood cells the major of in the PB (Fig. and at is more potent in HSPCs than In we that the SDF-1 levels not with the mobilization of HSPCs in patients and that normal and mobilized HSPCs of has been shown to HSPCs and to in the egress of HSPCs from extramedullary to with the activation of CC during we an of the in PB after G-CSF–induced which HSPC egress into the circulation in a chemotactic Furthermore, PB under steady-state conditions a significant of that a chemotactic gradient for we that retention of HSPCs in the BM is an process that the gradient continuously in the PB (Fig. our support the concept that the mobilization process is part of a more general immune response to or tissue injury that release of and HSPCs from hematopoietic that modulation of the CC could be an important to regulate the release of HSPC from their also have evidence similar to mobilization by the CXCR4 on activation of the mobilization in that not activate the of CC are C3 but is not as in mobilizing that are to the components of CC activation are In to activates the complement system at the C5 Our cell egress showed that granulocytes and monocytes the first of cells mobilized into the PB by by HSPCs. up the expression of and neutrophil that cleave and activate C5 in this BM-derived granulocyte and support the that C5 cleavage fragments and the of CC activation are required for mobilization of HSPCs (Fig. a antibody against C5 that a terminal complement is to lysis of in patients with and we can that this antibody would mobilization of to our such studies have been In recent there have been a number of on HSPC is as the that and firmly HSPC in the BM their proliferation and the of clinical HSPC transplantation on the ability of transplanted HSPCs to to the appropriate BM niche and to that to HSPC homing are place within 5 to and not later than days after occurs when IV HSPCs and with the endothelial cells of the BM, to the with to the stress by the and then the vascular along an SDF-1 gradient by osteoblasts in the endosteal niche (Fig. This homing gradient of SDF-1 is by small molecules, such as C3a, or released in the BM microenvironment after for transplantation by These small molecules increase the of HSPCs to the SDF-1 gradient by increasing of CXCR4 into membrane by up CXCR4 levels on HSPCs (PGE2), or by other mechanisms of of the interactions during HSPC for transplantation induces a proteolytic microenvironment (e.g., release of that activates the CC and leads to the of the In addition, the of the bioactive and phosphate increase in the BM after for transplantation. and are potent for HSPCs. Moreover, agents responses to SDF-1 (e.g., C3a, which through its receptor enhance the of CXCR4 into the of the cell membrane of HSPCs. All interactions to and of HSPCs in the BM niche. it during for transplantation in murine activates the CC in BM as by which C3a and C5a cleavage fragments in and by of in BM tissue (Fig. The of the CC in stem cell homing and engraftment is by the that and delayed hematopoietic after transplantation of the role of in HSPC we that its receptor also known as human is on HSPCs from human BM, and CB cells and on and progenitors (Fig. Moreover, is not a for cells, but it as well as migration of The presence of functional on human cells cells to better to the SDF-1 gradient and be retained within BM niches (Fig. shown that the of cells to an SDF-1 gradient in for in vivo studies we that are more to mobilization by G-CSF than suggesting that is in the retention of HSPCs in the BM niches and that of this axis egress of HSPC into the Moreover, to we showed that C3a and of HSPC to the SDF-1 which in BM after for A explanation for this is based on the that the signaling CXCR4 receptor is associated with (Fig. These membrane are rich in and which form a in a The are known to as on the cell surface and are more and to than other of the These are also sites for between various cell signaling For example, it has been recently that small such as and which are for engraftment of hematopoietic cells after transplantation, are associated with on Since the CXCR4 receptor is a its signaling ability is when CXCR4 is into membrane where it may better with signaling molecules, This of CXCR4 and in binding and activation of Thus, of C3 cleavage fragments in the BM microenvironment may act in as a to increase the of HSPCs to an SDF-1 In this is delayed In support of increases in the of C3a and in BM after promote homing of In addition, the C3 cleavage that is in the BM microenvironment HSPCs and increases their adhesion to Furthermore, evidence is to that the receptor is expressed by HSPCs and may be in their homing (Fig. transplantation of HSPCs into in a in neutrophil and PLT and a in the number of progenitors in the BM after transplantation. with murine blockage of on human CB cells by the their engraftment in results that the axis plays a role in the homing of HSPCs to the BM, first by enhancing to the SDF-1 gradient and, by modulating functions to have been that one of the strategies to be to improve engraftment is the ex vivo of HSPC transplantation with small molecules such as This is being in transplantation and where umbilical HSPCs are ex vivo with recombinant C3a for into the patients. In addition to C3 cleavage we that the cationic which is from granulocytes and BM is to the of murine and human HSPCs to an SDF-1 This indicates that the of HSPCs to the BM SDF-1 gradient could be by small molecules, as we with and Since the CC activated during mobilization of HSPCs and their via we that an mPB HSPCs that are by C1q, C3 cleavage and released from granulocytes in the leukapheresis This may mPB faster than BM or CB our recent that also poorly with HSPCs indicates that the could be in the homing of HSPCs (Fig. released in the of CC activation is in PB in two (Fig. The first MAC, that the of target cells, to cell lysis and However, another MAC, may bind to cell of receptor and not cells, but activates multiple signaling pathways and has on many cell to cellular such as and cell we and proposed that increases the homing responses of HSPC to and (Fig. A new is that that CC proteins play an important role in both the mobilization and the homing of HSPCs. These for clinical both ex vivo and in vivo that could improve the of HSPC transplantation. vivo of HSPCs with C3a the and is being in clinical The that have of to the to

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