Alma J. Nauta

Mesenchymal Stem Cells Inhibit Generation and Function of Both CD34+-Derived and Monocyte-Derived Dendritic Cells

Mesenchymal stem cells (MSCs) are not only able to evade the immune system, but they have also been demonstrated to exert profound immunosuppressive properties on T cell proliferation. However, their effect on the initiators of the immune response, the dendritic cells (DCs), are relatively unknown. In the present study, the effects of human MSCs on the differentiation and function of both CD34+-derived DCs and monocyte-derived DCs were investigated. The presence of MSCs during differentiation blocked the differentiation of CD14+CD1a− precursors into dermal/interstitial DCs, without affecting the generation of CD1a+ Langerhans cells. In line with these observations, MSCs also completely prevented the generation of immature DCs from monocytes. The inhibitory effect of MSCs on DC differentiation was dose dependent and resulted in both phenotypical and functional modifications, as demonstrated by a reduced expression of costimulatory molecules and hampered capacity to stimulate naive T cell proliferation. The inhibitory effect of MSCs was mediated via soluble factors. Taken together, these data demonstrate that MSCs, next to the antiproliferative effect on T cells, have a profound inhibitory effect on the generation and function of both CD34+-derived and monocyte-derived DCs, indicating that MSCs are able to modulate immune responses at multiple levels.

Sarcoma derived from cultured mesenchymal stem cells

To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole‐body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain‐specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon‐related genetic abnormality, and p53 was not overexpressed. Infusion of MSC‐derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.

Mannose‐binding lectin engagement with late apoptotic and necrotic cells

The serum opsonin mannose‐binding lectin (MBL) has been shown to be involved in the handling of apoptotic cells. However, at what stage in the process this happens and whether this mediates activation of complement is unknown. Cells rendered apoptotic or necrotic were incubated with purified MBL/MBL‐associated serine protease (MASP) complexes and assessed by flow cytometry and fluorescence microscopy. MBL bound specifically to late apoptotic cells, as well as to apoptotic blebs and to necrotic cells, but not to early apoptotic cells. Binding of MBL could be inhibited by EDTA as well as with an antibody against the CRD region. Addition of C1q, another serum opsonin involved in the handling of apoptotic cells, prior to MBL partly inhibited MBL binding to apoptotic cells and vice versa. MBL/MASP could initiate deposition of purified complement C4 on the target cells. However, addition of MBL/MASP to whole serum deficient for both C1q and MBL did not enhance deposition of C4, but MBL enhanced phagocytosis of apoptotic cells by macrophages. These results demonstrate that MBL interacts with structures exposed on cells rendered late apoptotic or necrotic and facilitates uptake by macrophages. Thus, MBL may promote non‐inflammatory sequestration of dying host cells.

Biochemical and functional characterization of the interaction between pentraxin 3 and C1q

Pentraxin 3 (PTX3) is a recently characterized member of the pentraxin family of acute‐phase proteins produced during inflammation. Classical short pentraxins, C‐reactive protein, and serum amyloid P component can bind to C1q and thereby activate the classical complement pathway. Since PTX3 can also bind C1q, the present study was designed to define the interaction between PTX3 and C1q and to examine the functional consequences of this interaction. A dose‐dependent binding of both C1q and the C1 complex to PTX3 was observed. Experiments with recombinant globular head domains of human C1q A, B, and C chains indicated that C1q interacts with PTX3 via its globular head region. Binding of C1q to immobilized PTX3 induced activation of the classical complement pathway as assessed by C4 deposition. Furthermore, PTX3 enhanced C1q binding and complement activation on apoptotic cells. However, in the fluid‐phase, pre‐incubation of PTX3 with C1q resulted in inhibition of complement activation by blocking the interaction of C1q with immunoglobulins. These results indicate that PTX3 can both inhibit and activate the classical complement pathway by binding C1q, depending on the way it is presented. PTX3 may therefore be involved in the regulation of the innate immune response.

Direct binding of C1q to apoptotic cells and cell blebs induces complement activation

Deficiency of early components of the classical pathway of complement, particularly C1q, predisposes to the development of systemic lupus erythematosus. Several studies have suggested an association between the classical complement pathway and the clearance of apoptotic cells. Mice with a targeted deletion of the C1q gene develop a lupus‐like renal disease, which is associated with the presence of multiple apoptotic bodies in the kidney. In the present study we demonstrate that highly purified C1q binds to apoptotic cells and isolated blebs derived from these apoptotic cells. Binding of C1q to apoptotic cells occurs via the globular heads of C1q and induces activation of the classical complement pathway, as shown by the deposition of C4 and C3 on the surface of these cells and on cell‐derived blebs. In addition, for the first time, we demonstrate that surface‐bound C1q is present on a subpopulation of microparticles isolated from human plasma. Taken together, these observations demonstrate that C1q binds directly to apoptotic cells and blebs derived therefrom and support a role for C1q, possibly in concert with C4 and C3, in the clearance of apoptotic cells and blebs by the phagocytic system.

Opsonization with C1q and Mannose-Binding Lectin Targets Apoptotic Cells to Dendritic Cells

Deficiencies of early components of the classical complement pathway, particularly C1q, are strongly associated with susceptibility to systemic lupus erythematosus. Recent data link this predisposal to autoimmunity to an inappropriate clearance of apoptotic cells, which could lead to a loss of self-tolerance. In the present study, we demonstrate that opsonization of apoptotic cells with C1q and mannose-binding lectin allows and facilitates their uptake not only by macrophages but also by human immature dendritic cells (DCs). Both C1q and mannose-binding lectin enhance the uptake of apoptotic cells by DCs in a dose-dependent way. The uptake of C1q-opsonized apoptotic cells, but not nonopsonized apoptotic cells, by DCs stimulated the production of IL-6, IL-10, and TNF-α, without an effect on IL-12p70. We conclude that these recognition molecules of the complement system do not sequester apoptotic cells from DCs, but rather promote their uptake by immature DCs. Therefore, we propose that early complement components support safe clearance of cellular debris by facilitating phagocytosis and possibly by immunomodulatory mechanisms, thus preventing autoimmunity.

Recognition and clearance of apoptotic cells: a role for complement and pentraxins

Apoptotic cells are specifically recognized and rapidly removed by professional phagocytes or neighboring cells by incompletely characterized mechanisms. Apoptotic cells are a potential source of autoantigens and, therefore, their efficient elimination, thus preventing unwanted immune reactions is essential. Accumulating evidence suggests that molecules of the innate immune system, including complement components and pentraxins, have a role in the removal of apoptotic cells. Therefore, it has been postulated that in situations with massive apoptosis, defective removal of apoptotic material, as a result of hampered opsonization by pentraxins and complement, can lead to the development of an autoimmune response. In this Review the potential role of complement and pentraxins in the clearance of apoptotic cells will be discussed.

Mini-review: A pivotal role for innate immunity in the clearance of apoptotic cells.

Apoptotic cells can be recognized and taken up by both macrophages and dendritic cells. Phagocytosis of apoptotic cells generally leads to active suppression of cytokine production by professional phagocytes. This is different from the response towards cells that die by necrosis, which induce a pro‐inflammatory cytokine profile. Uptake of apoptotic cells involves a large number of receptors and opsonins, which bind to cellular ligands exposed during the various stages of apoptotic cell death. Among the opsonins of apoptotic cells, complement factors, including C1q, and complement‐activating members of the pentraxin family play an important role. This is indicated by in vitro phagocytosis studies and supported by the susceptibility to systemic autoimmunity of carriersof genetic deficiencies for early complement proteins. The present review summarizes the role of molecules of innate immunity in the handling of apoptotic cells by macrophages and dendritic cells. It is proposed that C1q and other opsonins prevent autoimmunity and maintain self‐tolerance by supporting the efficient clearance of apoptotic material, as well as by actively modulating phagocyte function.

The membrane attack complex of complement induces caspase activation and apoptosis

Activation of the terminal pathway of the complement system leads to insertion of terminal complement complexes (C5b‐9) into the cell membrane, which may induce cytolysis. Recent data indicatethat the terminal complement pathway can also result in apoptosis in vivo. To further define the cell death pathway induced by complement, we examined induction of apoptosis by complement in vitro. Rat mesangial cells opsonized with a complement‐activating antibody and exposed to rat serum as a complement source underwent apoptotic cell death in a time‐ and dose‐dependent fashion, as demonstrated by membrane exposure of phosphatidylserine and fragmentation of nuclei. No significant apoptosis was detected in either cultures treated with C6‐deficient serum or in control cultures. The pan‐caspase‐inhibitor zVAD‐fmk inhibited complement‐induced apoptosis completely. In line with this observation, complement induced cleavage and activation of caspase 3. Importantly, cellular exposure to purified cytolytically inactive C5b‐9, in the absence of antibody and early complement components, also resulted into caspase activation and apoptosis. Together, these results indicate that C5b‐9 is involved in induction of apoptosis via a caspase‐dependent pathway. Apoptosis as a consequence of complement‐mediated cell damage may provide an explanation for the presence of apoptosis in inflammatory processes, for instance in hyperacute xenograft rejection.

Modulation of Immune Responses by Mesenchymal Stem Cells

Abstract:  Mesenchymal stem cells (MSCs) are multipotent progenitor cells and interest in MSC therapy has been raised by the observation that MSCs are able to modulate immune responses in vitro and in vivo. Here, we show that MSCs are not intrinsically immune privileged and are capable of inducing memory T cell responses following injection in vivo in immunocompetent hosts. After cotransplantation in recipients that have received sublethal irradiation, allogeneic MSCs can still induce an alloresponse that may result in graft rejection, suggesting that the immunogenicity of allogeneic MSCs are not fully prevented by a nonmyeloablative conditioning regimen. It is still unclear whether the immunogenicity of allogeneic MSCs is also preserved following a fully myeloablative conditioning regimen.