0 1992 Hanvood Academic Publishers GmbH
Leukemia and Lymphoma, Vol. 8, pp. 15-22 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
Bone Marrow Microenvironment and the Progression of Multiple Myeloma F. CALIGARIS-CAPPIO, M. G. GREGORETTI, F. MERICO, D. GOTTARDI, P. GHIA, G. PARVIS, and L. BERGUI Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione Clinica, Universita di Torino, Torino, Italy. (Received 25 February 1992)
The BM microenvironment in MM, in terms of adhesive features, is well organized to entrap circulating precursors with BM-seeking properties and is able to produce cytokines that offer them the optimal conditions for local growth and final differentiation. Likewise, the malignant B cell clone is equipped with adhesion molecules which enable the cell to establish close contacts with BM stromal cells. Furthermore a number of cytokines are released including IL-1/3 and M-CSF activating 1 BM stromal cells) to produce other cytokines, such as IL-6, that stimulate the proliferation of plasma cells. Finally, most cytokines produced locally, including IL-lj, TNF-/3, M-CSF, IL-3 and IL-6, also have OAF properties, explaining why the expansion of the B cell clone parallels the activation and numerical increase of the osteoclast population. KEY WORDS:
B lymphocytes plasma cells adhesion molecules
INTRODUCTION
cytokines
stromal cells
the distribution of normal plasma cells that are present not only in the BM, but also in the lamina propria of the gut and in different areas of secondary lymphoid organs such as lymph nodes and the spleen. The lamina propria of the intestine contains more Ig-producing cells than all other tissues in the body, yet it is never a site where M M develops (Ref. 3 and references therein). Among the plasma cell malignancies, only the rare a-chain disease is primarily localized in the gut. Likewise, the involvement of the spleen and/or lymph nodes is typical for Waldenstrom’s macroglobulinaemia but very unusual for MM3. These observations which fit in with the hypothesis that normal B cells can follow multiple pathways to give rise to Ig-producing cells, suggest that microenvironmental influences may be at least as important as lineage sequences in determining the fate of B cells in uiuo and favour the existence, within the MM BM, of some microenvironmental features that are essential for the growth of malignant plasma cell^^*^. The aim of this report is to review a series of recent
Plasma cells are the end effector cells of humoral immunity that originate from antigen (Ag)-driven B lymphocyte activation, proliferation and differentiation. The most frequent plasma cell malignancy is multiple myeloma (MM), a neoplasm characterized by the accumulation of plasma cells that secrete monoclonal immunoglobulins (Ig) and cause typical osteolytic lesions’. Though MM has a clear position in the hierarchical framework of normal B cell differentiation which is used to classify B cell malignancies2, it’s tissue distribution raises two major questions; one is which is the normal counterpart of MM? while the other relates to which mechanisms regulate its progression? MM malignant plasma cells are found only within the bone marrow (BM) where they are disseminated from the earliest recognizable stage of the disease’. This localization contrasts with Address for correspondence: F. Caligaris-Cappio, Cattedra di Immunologia Clinica, Via Genova 3, 10126 Torino, Italy. 15
16
F. CALIGARIS-CAPPIO
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
findings that may provide a clue in understanding why MM is uniquely a BM malignancy. In this respect we will attempt to relate the heterogeneity of normal plasma cells to some distinctive features of MM malignant plasma cells and the properties of BM microenvironment that may contribute to the disease progression will also be discussed.
THE HETEROGENEITY OF NORMAL PLASMA CELLS Different microenvironments where normal B cells can be activated to give rise to plasma cells exist in different anatomical sites, (Ref. 4 and references therein). These include macrophage-rich areas, such as the red pulp of the spleen, areas of secondary lymphoid tissues with many T lymphocytes and interdigitating cells and secondary lymphoid follicles where B cell activation is promoted by Ag associated with follicular dendritic cells. These different microenvironments provide the ground where T-cell (in)dependent primary and secondary antibody (Ab) responses also take place. The properties of plasma cells, including their Ig isotype and their individual life span, vary according to the site where they are generated4. Plasma cells generated in macrophagerich areas after T-cell independent Ag challenge secrete IgM and are short-lived, while secondary T-cell dependent Ab response leads to the production of IgG- or IgA-secreting plasma cells with a life span of few weeks (reviewed in Ref. 4). The BM is a major site where IgG and IgA are produced in T-cell dependent secondary immune responses5. Nevertheless the steps of Ag processing and presentation that lead to the generation of IgG and IgA plasma cells only occur in secondary lymphoid follicles, which are distant from the BM6. The missing link is provided by the migration of stimulated cells (plasma cell precursors) from secondary lymphoid organs to the BM a few days after the antigenic challenge6*’. These precursors must have specific traffic committments as only those generated in the follicles of the spleen or peripheral lymph nodes migrate to the BM. while the precursors derived from the follicles of Peyer’s patches or mesenteric lymph nodes migrate to the lamina propria of the gut*. Thus it is reasonable to assume that BM-seeking plasma cell precursors receive a differentiation signal at the time they leave the blood in order to come into contact with the BM stroma6.
ef
al.
The heterogeneity of normal plasma cells may be used to interpret two aspects of this disease. Firstly, the Ig isotypes of MM plasma cells are generally IgG or IgA, while IgM-producing MM are indeed exceptional. Secondly, ample experimental evidence suggests the existence of malignant plasma cell precursors circulating in the peripheral blood of MM patients9-14. These findings, taken together, lead to the hypothesis that the normal cell population from which MM may arise, is represented by plasma cell precursors generated in peripheral lymphoid organs during secondary T-cell dependent Ab response, that are programmed to home to the BM and are committed to differentiate once they come into close association with the BM rnicr~environment~*~*’~. The implication is that the progression of MM is due to the interactions between the malignant B cell clone and the BM microenvironment.Thus it is appropriate to analyse the main properties of these interacting cell populations.
MM MALIGNANT PLASMA CELLS: GROWTH FACTOR PRODUCTION AND REQUIREMENTS MM malignant plasma cells are not inert vehicles of monoclonal Ig, but produce several cytokines which are able to influence different cell populations. These include interleukin (IL)-l/3, tumor necrosis factor (TNF)-fl and a functionally active truncated version of the monocyte-macrophage colony stimulating factor (M-CSF) (15-18). It is still unclear whether fresh MM plasma cells may produce IL-6, despite the fact that a minority of human MM cell lines autonomously produce small amounts of IL-619-”. IL-l/3 and TNF-/I are osteoclast-activating factors (OAF; Ref. 17,22,23) while M-CSF and IL-6 play an important role in promoting the differentiation of osteoclasts supported by stromal cell^^^^^^. IL-1fl and IL-6 promote B cell proliferation and differentiation and also activate stromal and accessory In uitro, the growth of human MM cell lines can be facilitated by supplementing the culture medium with IL-6 or is dependent on IL-6 producing feeder cells like fibroblasts, gIial cells or macrophages”. IL-6, a potent growth factor for murine hybridomas and plasmacytomas26*28, is, at present, the most relevant growth factor known for human MM26*29.About &60% of MM cell lines increase 3H-Tdr incorporation after IL-6 stimulation and a proliferation
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
BONE MARROW MICROENVIRONMENT IN MYELOMA
response to IL-6 also occurs in fresh MM samp l e ~ ~ ~Other * ~ cytokines, ~ * ~ ~ .such as granulocyte macrophage (GM)-CSF, IL-1, IL-3 and IL-5, frequently in synergy with IL-6, may increase the 3H-Tdr incorporation of purified MM plasma cells cultured Finally, an IL-6-like activity affecting in plasmacytomas has been shown by the recently cloned IL-1 132. The in vitro-active cytokines play a role in vivo as well as seen by the presence of high levels of IL-6 in the sera of patients with aggressive or progressive MM33and by the therapeutic effect of the infusion of antil-IL-6 Abs in patients with plasma cell leukemia34. IL-6 is produced by activated T cells, fibroblasts and accessory cells, including BM stromal cells26,IL-1 by a variety of cells with accessory function23;IL-3 and IL-5 by activated T lymphocyte^^'*^^ and IL-11 is produced by BM stromal cell lines32. These data indicate that a number of cells, including accessory cells and T lymphocytes, are potentially involved in the growth of the malignant cell clone through mutually interacting paracrine loops.
17
myeloid progenitors is yet to be defined. Long-term lymphoid cultures from murine BM were established by Whitlock and Witte and allowed for the selective growth of B lineage cells4’. In mice, the WhitlockWitte system has allowed a detailed investigation of the stromal cells and cytokines that govern B cell development within the marrow (reviewed in Ref. 39). Informations from these studies have been extrapolated to humans, as until now a human equivalent of the murine Whitlock-Witte system has not yet been properly established. The recent production of a monoclonal antibody (MoAb) that identifies stromal cell precursors in human BM (STRO-1)41 may help in the future in dissecting the functional heterogeneity of stromal cell precursors and in understanding which particular elements favour the development of B lineage precursors.
BM STROMAL CELLS IN PATIENTS WITH MM
We have recently established in uitro cultures of stromal cells from the BM of patients with MM4*. After 7-8 weeks of culturing methylcelluloseBONE MARROW MICROENVIRONMENT separated MM BM nucleated cells in Dulbecco The BM has been studied mainly as an hematopoietic medium and 5% horse serum at 37°C in 5 % C 0 2 , a environment3’, however knowledge of which features population of adherent cells is obtained (Figure 1). facilitate its function as a major Ab producing organ These adherent cells form a confluent meshwork of intertwining elements that may be used as a feeder in man, are still in their infancy. Anatomically, the BM microenvironment consists layer for autologous peripheral blood mononuclear of a properly organized meshwork of cells, collectively cells (PBMC). Within MM BM stromal cells three distinct named “stromal cells”38. The definition “stromal cells’’ refers to an heterogeneous population of populations are observed. The vast majority of cells adherent elements which include fibroblasts, macro- have a fibroblast-like morphology (Figure la) and phages, adipocytes, endothelial cells, reticular cells, produce their own extracellular matrix by synthesizadventitial cells which are loosely distributed around ing fibronectin, laminin and collagen type IV. These BM sinusoids where they produce extracellular matrix elements are weakly CDlO +, CD54 +, express the glycoproteins and a large array of c y t o k i n e ~ ~ ~integrin . ~ ~ . PI diffusely on their surface and concentrate Functionally, the BM microenvironment is a complex the integrin P3 in the adhesion plaques. Scattered system that supports and regulates the sustained among these fibroblasts, several macrophages are seen production of mature lymphohemopoietic cells, which are CD54+, CD56+, express the integrin including B lymphocyte^^'*^^. The influence of diffusely on their surface and the integrin f12 in the stromal cells in the process of cell differentiation is podosomes. Within this network occasional giant exerted through multiple molecular contacts which multinucleated cells are evident that exhibit osteoclast are mediated by adhesion molecules and result in the features. (Figures l b and c). These osteoclast-like cells local production of both stimulatory and inhibitory are CD54 CD56 +, express surface PI integrin and concentrate P3 integrin in their podosomes. The cytokines39. The individual significance of the different stromal number of osteoclasts growing in uitro is highly cell components in supporting the development of B variable and directly relates3 to the severity of the cell progenitors as compared to the development of disease staged according to Durie and Salmon43.
+,
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
Figure 1 Bone marrow (BM) stromal cells in multiple myeloma (MM).(A): fibroblast-like cells (revealed by Ruoresceinated phalloidin that selectively binds to F-actin) form the bulk of stromal cells in the BM of MM. Within the meshwork of fibroblast-like cells lie large multi-nucleated cells. (See Colour Plate I at the back of this publication.)
Figure 1B These cells have all the features of osteoclasts, including the organization of F-actin into podosomes located in a ring-like fashion at the periphery of the cell. (See Colour Plate I1 at the back of this publication)
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
BONE MARROW MICROENVIRONMENT IN MYELOMA
19
Figure 1C Double staining with fluoresceinated phalloidin (green) and rhodaminated anti-pl integrin (red). (See colour Plate 111 at the back of this publication.)
IL-1B, high levels of IL-6, IL-7 and IL-8 can also be detected in the supernatants of cultures where MM BM stromal cells are growing, while IL-2 and TNF-a are undetectable (Ref. 3 and manuscript in preparation). MM BM STROMAL CELLS AND THE PROGRESSION OF THE DISEASE When BM stromal cells are used as feeder layers for autologous PBMC two events are observed after 3 weeks of co-culture: (a) a monoclonal B lineage cell population develops42 and these cells grow tightly adherent to the stromal cell layer; (b) the osteoclastlike cell population is significantly expanded. Both events occur in association with a striking increase of IL-6 concentration in the culture supernatant, where IL-3 levels also become m e a ~ u r a b l eThese ~ ~ . observations indicate that BM stromal cells in MM are fully equipped with a large series of adhesion and extracellular matrix molecules; produce several cytokines that are known to play a crucial role in the
evolution of the disease; support the growth of peripheral blood-borne monoclonal B-cell precursors and are involved in the expansion of the osteoclast compartment. It is now well known that cell-cell interactions and cell adhesion to extracellular matrix proteins are mediated by the integrin family of cell surface receptors44.Furthermore, extracellular matrix proteins secreted by fibroblast-like cells may provide anchorage sites where cells are selectively exposed to locally released growth MM plasma cells express adhesion molecules on their surface like H-CAM (CD44), N-CAM (CD56) and ICAM-I (CD54)47-49.These data have been confirmed in a study on BM cells from our series of MM patients (manuscript in preparation) which has also shown that monoclonal Ig lymphoid-like cells are CD54 + , CD56 - , CD18 . Thus, several adhesion molecules are present on the surface.of the different elements of the malignant B cell clone. Conceivably these molecules, by interacting with the homologous ligands in the BM microenvironment, may allow malignant B cells to be entrapped within the BM stromal cell web. Thereby, they would be exposed to cytokines,
+ +
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
20
F. CALIGARIS-CAPPIO et al.
like IL-6, which promote B cell proliferation and differentiati~n~~.~'. In addition, circulating T cells that reach the marrow may also become activated to produce IL-3 and GM-CSF via locally released IL-7. Finally, osteoclast precursors could also be stimulated by a whole range of cytokines with OAF properties, like IL-lfl, TNF-fl, M-CSF, IL-3 and IL-617.22.24.25.50.51 On these bases, an attractive explanation for the progression of MM is to postulate a self-maintaining series of mutual interactions between the malignant B cell clone and the BM micr~environment~. The expansion of the malignant B cell clone would lead to the production of ever-increasing amounts of cytokines which are able to recruit and activate several microenvironmental cells including osteoclasts and accessory cells. Activated accessory cells, in turn, would produce IL-6 (and perhaps IL-11) and thus further enhance the malignant B cell clone pool.
BM STROMAL CELLS IN MGUS AND NORMAL DONORS If such a hypothesis is correct, BM stromal cells from MM would be expected to be in a different state of activation when compared to BM stromal cells derived from MGUS and normal donors. In our studies we have experienced real difficulties in obtaining stromal cell cultures from the BM of MGUS and normal donors age-matched with M M patients. The cultures from these subjects are not confluent and osteoclasts are not evident. The rare cells which grow out are fibroblast-like elements and sparse macrophages. Furthermore these stromal cells do not support the growth of autologous B cells and IL-6 levels in the culture supernatants are very low. Our failure with normal donors may simply reflect an age-related feature as we have obtained stromal cell cultures from the BM of younger individuals, less than 30yr of age, (manuscript in preparation). In these cultures, fibroblast-like cells grow actively, easily reach confluence and are interspersed with macrophages. However, osteoclast-like cells are very rarely seen and IL-6 levels are significantly lower than those observed in MM BM cultures (Table 1). These findings lead us to conclude that in the BM of M M a number of environmental cells, that are normally quiescent particularly in a certain age range, are in fact activated. It is not unreasonable to consider the activated state of stromal cells in MM as a direct consequence of host accessory cell-activating cyto-
Table 1 Interleukin 6 in the supernatants of BM stromal cell cultures MM MGUS ND
14/18" 415 013
+ +
2.1 1.6 ng/ml (0.5-3.7) 1.1 1.2 ng/ml (0.2-2.3)
Leukemia and Lymphoma, Vol. 8, pp. 15-22 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
Bone Marrow Microenvironment and the Progression of Multiple Myeloma F. CALIGARIS-CAPPIO, M. G. GREGORETTI, F. MERICO, D. GOTTARDI, P. GHIA, G. PARVIS, and L. BERGUI Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione Clinica, Universita di Torino, Torino, Italy. (Received 25 February 1992)
The BM microenvironment in MM, in terms of adhesive features, is well organized to entrap circulating precursors with BM-seeking properties and is able to produce cytokines that offer them the optimal conditions for local growth and final differentiation. Likewise, the malignant B cell clone is equipped with adhesion molecules which enable the cell to establish close contacts with BM stromal cells. Furthermore a number of cytokines are released including IL-1/3 and M-CSF activating 1 BM stromal cells) to produce other cytokines, such as IL-6, that stimulate the proliferation of plasma cells. Finally, most cytokines produced locally, including IL-lj, TNF-/3, M-CSF, IL-3 and IL-6, also have OAF properties, explaining why the expansion of the B cell clone parallels the activation and numerical increase of the osteoclast population. KEY WORDS:
B lymphocytes plasma cells adhesion molecules
INTRODUCTION
cytokines
stromal cells
the distribution of normal plasma cells that are present not only in the BM, but also in the lamina propria of the gut and in different areas of secondary lymphoid organs such as lymph nodes and the spleen. The lamina propria of the intestine contains more Ig-producing cells than all other tissues in the body, yet it is never a site where M M develops (Ref. 3 and references therein). Among the plasma cell malignancies, only the rare a-chain disease is primarily localized in the gut. Likewise, the involvement of the spleen and/or lymph nodes is typical for Waldenstrom’s macroglobulinaemia but very unusual for MM3. These observations which fit in with the hypothesis that normal B cells can follow multiple pathways to give rise to Ig-producing cells, suggest that microenvironmental influences may be at least as important as lineage sequences in determining the fate of B cells in uiuo and favour the existence, within the MM BM, of some microenvironmental features that are essential for the growth of malignant plasma cell^^*^. The aim of this report is to review a series of recent
Plasma cells are the end effector cells of humoral immunity that originate from antigen (Ag)-driven B lymphocyte activation, proliferation and differentiation. The most frequent plasma cell malignancy is multiple myeloma (MM), a neoplasm characterized by the accumulation of plasma cells that secrete monoclonal immunoglobulins (Ig) and cause typical osteolytic lesions’. Though MM has a clear position in the hierarchical framework of normal B cell differentiation which is used to classify B cell malignancies2, it’s tissue distribution raises two major questions; one is which is the normal counterpart of MM? while the other relates to which mechanisms regulate its progression? MM malignant plasma cells are found only within the bone marrow (BM) where they are disseminated from the earliest recognizable stage of the disease’. This localization contrasts with Address for correspondence: F. Caligaris-Cappio, Cattedra di Immunologia Clinica, Via Genova 3, 10126 Torino, Italy. 15
16
F. CALIGARIS-CAPPIO
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
findings that may provide a clue in understanding why MM is uniquely a BM malignancy. In this respect we will attempt to relate the heterogeneity of normal plasma cells to some distinctive features of MM malignant plasma cells and the properties of BM microenvironment that may contribute to the disease progression will also be discussed.
THE HETEROGENEITY OF NORMAL PLASMA CELLS Different microenvironments where normal B cells can be activated to give rise to plasma cells exist in different anatomical sites, (Ref. 4 and references therein). These include macrophage-rich areas, such as the red pulp of the spleen, areas of secondary lymphoid tissues with many T lymphocytes and interdigitating cells and secondary lymphoid follicles where B cell activation is promoted by Ag associated with follicular dendritic cells. These different microenvironments provide the ground where T-cell (in)dependent primary and secondary antibody (Ab) responses also take place. The properties of plasma cells, including their Ig isotype and their individual life span, vary according to the site where they are generated4. Plasma cells generated in macrophagerich areas after T-cell independent Ag challenge secrete IgM and are short-lived, while secondary T-cell dependent Ab response leads to the production of IgG- or IgA-secreting plasma cells with a life span of few weeks (reviewed in Ref. 4). The BM is a major site where IgG and IgA are produced in T-cell dependent secondary immune responses5. Nevertheless the steps of Ag processing and presentation that lead to the generation of IgG and IgA plasma cells only occur in secondary lymphoid follicles, which are distant from the BM6. The missing link is provided by the migration of stimulated cells (plasma cell precursors) from secondary lymphoid organs to the BM a few days after the antigenic challenge6*’. These precursors must have specific traffic committments as only those generated in the follicles of the spleen or peripheral lymph nodes migrate to the BM. while the precursors derived from the follicles of Peyer’s patches or mesenteric lymph nodes migrate to the lamina propria of the gut*. Thus it is reasonable to assume that BM-seeking plasma cell precursors receive a differentiation signal at the time they leave the blood in order to come into contact with the BM stroma6.
ef
al.
The heterogeneity of normal plasma cells may be used to interpret two aspects of this disease. Firstly, the Ig isotypes of MM plasma cells are generally IgG or IgA, while IgM-producing MM are indeed exceptional. Secondly, ample experimental evidence suggests the existence of malignant plasma cell precursors circulating in the peripheral blood of MM patients9-14. These findings, taken together, lead to the hypothesis that the normal cell population from which MM may arise, is represented by plasma cell precursors generated in peripheral lymphoid organs during secondary T-cell dependent Ab response, that are programmed to home to the BM and are committed to differentiate once they come into close association with the BM rnicr~environment~*~*’~. The implication is that the progression of MM is due to the interactions between the malignant B cell clone and the BM microenvironment.Thus it is appropriate to analyse the main properties of these interacting cell populations.
MM MALIGNANT PLASMA CELLS: GROWTH FACTOR PRODUCTION AND REQUIREMENTS MM malignant plasma cells are not inert vehicles of monoclonal Ig, but produce several cytokines which are able to influence different cell populations. These include interleukin (IL)-l/3, tumor necrosis factor (TNF)-fl and a functionally active truncated version of the monocyte-macrophage colony stimulating factor (M-CSF) (15-18). It is still unclear whether fresh MM plasma cells may produce IL-6, despite the fact that a minority of human MM cell lines autonomously produce small amounts of IL-619-”. IL-l/3 and TNF-/I are osteoclast-activating factors (OAF; Ref. 17,22,23) while M-CSF and IL-6 play an important role in promoting the differentiation of osteoclasts supported by stromal cell^^^^^^. IL-1fl and IL-6 promote B cell proliferation and differentiation and also activate stromal and accessory In uitro, the growth of human MM cell lines can be facilitated by supplementing the culture medium with IL-6 or is dependent on IL-6 producing feeder cells like fibroblasts, gIial cells or macrophages”. IL-6, a potent growth factor for murine hybridomas and plasmacytomas26*28, is, at present, the most relevant growth factor known for human MM26*29.About &60% of MM cell lines increase 3H-Tdr incorporation after IL-6 stimulation and a proliferation
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
BONE MARROW MICROENVIRONMENT IN MYELOMA
response to IL-6 also occurs in fresh MM samp l e ~ ~ ~Other * ~ cytokines, ~ * ~ ~ .such as granulocyte macrophage (GM)-CSF, IL-1, IL-3 and IL-5, frequently in synergy with IL-6, may increase the 3H-Tdr incorporation of purified MM plasma cells cultured Finally, an IL-6-like activity affecting in plasmacytomas has been shown by the recently cloned IL-1 132. The in vitro-active cytokines play a role in vivo as well as seen by the presence of high levels of IL-6 in the sera of patients with aggressive or progressive MM33and by the therapeutic effect of the infusion of antil-IL-6 Abs in patients with plasma cell leukemia34. IL-6 is produced by activated T cells, fibroblasts and accessory cells, including BM stromal cells26,IL-1 by a variety of cells with accessory function23;IL-3 and IL-5 by activated T lymphocyte^^'*^^ and IL-11 is produced by BM stromal cell lines32. These data indicate that a number of cells, including accessory cells and T lymphocytes, are potentially involved in the growth of the malignant cell clone through mutually interacting paracrine loops.
17
myeloid progenitors is yet to be defined. Long-term lymphoid cultures from murine BM were established by Whitlock and Witte and allowed for the selective growth of B lineage cells4’. In mice, the WhitlockWitte system has allowed a detailed investigation of the stromal cells and cytokines that govern B cell development within the marrow (reviewed in Ref. 39). Informations from these studies have been extrapolated to humans, as until now a human equivalent of the murine Whitlock-Witte system has not yet been properly established. The recent production of a monoclonal antibody (MoAb) that identifies stromal cell precursors in human BM (STRO-1)41 may help in the future in dissecting the functional heterogeneity of stromal cell precursors and in understanding which particular elements favour the development of B lineage precursors.
BM STROMAL CELLS IN PATIENTS WITH MM
We have recently established in uitro cultures of stromal cells from the BM of patients with MM4*. After 7-8 weeks of culturing methylcelluloseBONE MARROW MICROENVIRONMENT separated MM BM nucleated cells in Dulbecco The BM has been studied mainly as an hematopoietic medium and 5% horse serum at 37°C in 5 % C 0 2 , a environment3’, however knowledge of which features population of adherent cells is obtained (Figure 1). facilitate its function as a major Ab producing organ These adherent cells form a confluent meshwork of intertwining elements that may be used as a feeder in man, are still in their infancy. Anatomically, the BM microenvironment consists layer for autologous peripheral blood mononuclear of a properly organized meshwork of cells, collectively cells (PBMC). Within MM BM stromal cells three distinct named “stromal cells”38. The definition “stromal cells’’ refers to an heterogeneous population of populations are observed. The vast majority of cells adherent elements which include fibroblasts, macro- have a fibroblast-like morphology (Figure la) and phages, adipocytes, endothelial cells, reticular cells, produce their own extracellular matrix by synthesizadventitial cells which are loosely distributed around ing fibronectin, laminin and collagen type IV. These BM sinusoids where they produce extracellular matrix elements are weakly CDlO +, CD54 +, express the glycoproteins and a large array of c y t o k i n e ~ ~ ~integrin . ~ ~ . PI diffusely on their surface and concentrate Functionally, the BM microenvironment is a complex the integrin P3 in the adhesion plaques. Scattered system that supports and regulates the sustained among these fibroblasts, several macrophages are seen production of mature lymphohemopoietic cells, which are CD54+, CD56+, express the integrin including B lymphocyte^^'*^^. The influence of diffusely on their surface and the integrin f12 in the stromal cells in the process of cell differentiation is podosomes. Within this network occasional giant exerted through multiple molecular contacts which multinucleated cells are evident that exhibit osteoclast are mediated by adhesion molecules and result in the features. (Figures l b and c). These osteoclast-like cells local production of both stimulatory and inhibitory are CD54 CD56 +, express surface PI integrin and concentrate P3 integrin in their podosomes. The cytokines39. The individual significance of the different stromal number of osteoclasts growing in uitro is highly cell components in supporting the development of B variable and directly relates3 to the severity of the cell progenitors as compared to the development of disease staged according to Durie and Salmon43.
+,
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
Figure 1 Bone marrow (BM) stromal cells in multiple myeloma (MM).(A): fibroblast-like cells (revealed by Ruoresceinated phalloidin that selectively binds to F-actin) form the bulk of stromal cells in the BM of MM. Within the meshwork of fibroblast-like cells lie large multi-nucleated cells. (See Colour Plate I at the back of this publication.)
Figure 1B These cells have all the features of osteoclasts, including the organization of F-actin into podosomes located in a ring-like fashion at the periphery of the cell. (See Colour Plate I1 at the back of this publication)
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
BONE MARROW MICROENVIRONMENT IN MYELOMA
19
Figure 1C Double staining with fluoresceinated phalloidin (green) and rhodaminated anti-pl integrin (red). (See colour Plate 111 at the back of this publication.)
IL-1B, high levels of IL-6, IL-7 and IL-8 can also be detected in the supernatants of cultures where MM BM stromal cells are growing, while IL-2 and TNF-a are undetectable (Ref. 3 and manuscript in preparation). MM BM STROMAL CELLS AND THE PROGRESSION OF THE DISEASE When BM stromal cells are used as feeder layers for autologous PBMC two events are observed after 3 weeks of co-culture: (a) a monoclonal B lineage cell population develops42 and these cells grow tightly adherent to the stromal cell layer; (b) the osteoclastlike cell population is significantly expanded. Both events occur in association with a striking increase of IL-6 concentration in the culture supernatant, where IL-3 levels also become m e a ~ u r a b l eThese ~ ~ . observations indicate that BM stromal cells in MM are fully equipped with a large series of adhesion and extracellular matrix molecules; produce several cytokines that are known to play a crucial role in the
evolution of the disease; support the growth of peripheral blood-borne monoclonal B-cell precursors and are involved in the expansion of the osteoclast compartment. It is now well known that cell-cell interactions and cell adhesion to extracellular matrix proteins are mediated by the integrin family of cell surface receptors44.Furthermore, extracellular matrix proteins secreted by fibroblast-like cells may provide anchorage sites where cells are selectively exposed to locally released growth MM plasma cells express adhesion molecules on their surface like H-CAM (CD44), N-CAM (CD56) and ICAM-I (CD54)47-49.These data have been confirmed in a study on BM cells from our series of MM patients (manuscript in preparation) which has also shown that monoclonal Ig lymphoid-like cells are CD54 + , CD56 - , CD18 . Thus, several adhesion molecules are present on the surface.of the different elements of the malignant B cell clone. Conceivably these molecules, by interacting with the homologous ligands in the BM microenvironment, may allow malignant B cells to be entrapped within the BM stromal cell web. Thereby, they would be exposed to cytokines,
+ +
Leuk Lymphoma Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
20
F. CALIGARIS-CAPPIO et al.
like IL-6, which promote B cell proliferation and differentiati~n~~.~'. In addition, circulating T cells that reach the marrow may also become activated to produce IL-3 and GM-CSF via locally released IL-7. Finally, osteoclast precursors could also be stimulated by a whole range of cytokines with OAF properties, like IL-lfl, TNF-fl, M-CSF, IL-3 and IL-617.22.24.25.50.51 On these bases, an attractive explanation for the progression of MM is to postulate a self-maintaining series of mutual interactions between the malignant B cell clone and the BM micr~environment~. The expansion of the malignant B cell clone would lead to the production of ever-increasing amounts of cytokines which are able to recruit and activate several microenvironmental cells including osteoclasts and accessory cells. Activated accessory cells, in turn, would produce IL-6 (and perhaps IL-11) and thus further enhance the malignant B cell clone pool.
BM STROMAL CELLS IN MGUS AND NORMAL DONORS If such a hypothesis is correct, BM stromal cells from MM would be expected to be in a different state of activation when compared to BM stromal cells derived from MGUS and normal donors. In our studies we have experienced real difficulties in obtaining stromal cell cultures from the BM of MGUS and normal donors age-matched with M M patients. The cultures from these subjects are not confluent and osteoclasts are not evident. The rare cells which grow out are fibroblast-like elements and sparse macrophages. Furthermore these stromal cells do not support the growth of autologous B cells and IL-6 levels in the culture supernatants are very low. Our failure with normal donors may simply reflect an age-related feature as we have obtained stromal cell cultures from the BM of younger individuals, less than 30yr of age, (manuscript in preparation). In these cultures, fibroblast-like cells grow actively, easily reach confluence and are interspersed with macrophages. However, osteoclast-like cells are very rarely seen and IL-6 levels are significantly lower than those observed in MM BM cultures (Table 1). These findings lead us to conclude that in the BM of M M a number of environmental cells, that are normally quiescent particularly in a certain age range, are in fact activated. It is not unreasonable to consider the activated state of stromal cells in MM as a direct consequence of host accessory cell-activating cyto-
Table 1 Interleukin 6 in the supernatants of BM stromal cell cultures MM MGUS ND
14/18" 415 013
+ +
2.1 1.6 ng/ml (0.5-3.7) 1.1 1.2 ng/ml (0.2-2.3)
