tgf-β and megakaryocytes in the pathogenesis of myelofibrosis in myeloproliferative disorders

Leukemia and Lymphoma, Vol. 20, pp. 3 9 4 4 Reprints available directly from the publisher Photocopying permitted by license only

0 1995 Harwood Academic Publishers GmbH Printed in Singapore

TGF- p and Megakaryocytes in the Pathogenesis of Myelofibrosis in Myeloproliferative Disorders

MARIE-CLAIRE MARTYRE

Unit6 365 INSERM "interferons er Cyrokines” Institut Curie, Section de Biologie, Pavillon Pasteur 26 rue d‘Ulm, 75231 Paris c6dex 05 France

(Received February 3, 1995)

Myeloproliferative disorders are clonal disorders of the hematopoietic stem cell and comprise a spec- trum of more or less well-defined clinical entities: polycythaemia Vera, chronic myeloid leukemia, essential thrombocythaemia, and agnogenic myeloid metaplasia. Myelofibrosis, which contributes substantially to the impaired hematopoiesis, is commonly observed in myeloproliferative disorders but it represents the criterion of agnogenic myeloid metaplasia also termed idiopathic myelofibrosis. Although progress has been made in the elucidation of the pathogenesis of myelofibrosis, it still remains unclear. The aim of this review is to adress the new insights that outline the potential role of TGF-P in the promotion of myelofibrosis, through its release from megakaryocytes/platelets, partic- ularly in agnogenic myeloid metaplasia.

KEY WORDS: mvelofibrosis mvelomoliferative disorders megakqocytes - . TGF-P

The abnormal deposition of collagen in bone marrow is known as myelofibrosis. It was first described by Heuck in 1879 in two patients with severe bone marrow fibrosis and extramedullary hematopoiesis in the liver and spleen, and regarded as a sign of progression in leukemias.’ Since then, myelofibrosis has been recognized to occur during the course of myeloproliferative disorders (MPD) such as chronic myeloid leukemia (CML) and polycythaemia Vera (PV), in which it represents an advanced stage of the disease.2 In contrast, myelofibrosis is the criterion of the disease formerly described by Heuck, which was reported under a variety of names refering to particular clinical fea- tures of the disease. This chronic neoplastic disorder, clas- sified by Dameshek3 as a MPD, is now usually termed agnogenic myeloid metaplasia or idiopathic myelofibrosis with myeloid metaplasia (IMF); actually, in this disease, for unknown reasons, connective tissue progressively replaced hematopoietic bone marrow cells; besides bone marrow fibrosis, idiopathic myelofibrosis is characterized

Address for correspondence: Prof. Marie-Claire Martyr& Unit6 365 MSERM, Institut Curie, Pavillon Pasteur, 26 Rue d’Ulm, 7523 1 Pans, Cedex 05, France.

by extramedullary hemopoiesis, splenomegaly and a leu- coerythroblastic blood picture.4

Although progress has been made in the elucidation of its pathophysiology, the prognosis remains poor when compared to other MPD.5 The origin of the disease still remains unknown, however several lines of evidence suggested that the primary defect resides in the hematopoietic series, at the level of the pluripotent stem cell, and that the bone marrow fibrosis is a secondary reactive process.

CLONALITY OF HEMATOPOIESIS AND REACTIVE FIBROBLAST PROLIFERATION

The hypothesis of a clonal expansion of hematopoietic stem cell was first supported by findings of Kahn6 confirmed by Jacobson et al.7 Using G6PD isoenzyme analysis in a heterozygous female patient, they demonstrated that the erythrocytes, granulocytes and platelets were de- rivedfrom acommon hematopoietic stemcell, that is monoclonal, whereas fibroblast proliferation was polyclonal. These observations were subsequently confirmed by studies of fibroblast characteristics and proliferation kinetics,* the evidence of N-ras gene mutation in all three

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40 M. MARTYF&

hematopoietic lineages9 and more recently, by the cytogenetic analysis of bone marrow fibroblasts and blood cells from peripheral blood or bone marrow; 10 further, using X-linked restriction length polymorphism, mono- clonality of granulocytes or total bone marrow cells could be demonstrated in heterozygous female patients at various stages of agnogenic myeloid metaplasia.ll The finding of increased numbers of multilineage (CFU-GEMM) and committed (CFU-GM, BFU-E and CFU-MK) circulating progenitors in patients with agnogenic myeloid metaplasialz and the frequent transformations in acute leukemia also provide evidence that the primary defect may reside in the pluripotent stem cell; the reactive nature of the fibroblast proliferation is subsequently supported by data stating that the physical characteristics of myeloproliferative bone marrow fibroblasts appear identical to those of normal donors8 and by the report of resolution of bone marrow fibrosis following allogenic bone marrow transplantation. 13

ROLE OF MEGAKARYOCYTES AND GROWTH FACTORS

Megakaryoc y tes

Although probably of reactive origin, the pathogenesis of the myelofibrotic stroma tissue is still unclear; however several observations, in myeloproliferative disorders, led to the concept that the megakaryocytic lineage may play a pivotal role in its production. Indeed, there are convinc- ing data that favored the hypothesized role of megakaryocytes in the development of myelofibrosis, i) the megakaryocytic hyperplasia with dysplastic or necrotic megakaryocytes, ii) such an hyperplasia being often associated with the fibrotic tissue, i.e., collagen deposition and fibroblast proliferation,14 iii) the increased number of circulating megakaryocytes and their progenitors,IZ iv) the possible transformation in acute megakaryoblastic leukemia15 and the existence of myelofibrosis in the rare Grey platelet syndrome. l 6

By showing that megakaryocyte homogenates were capable to stimulate the proliferation of medullary fibroblasts, Castro-Malaspina et al. provided tangible evidence in support of the critical role of megakaryocytes and a growth factor synthesized by these cells and stored in platelets, namely the platelet-derived growth factor (PDGF), in the pathogenesis of myelofibrosis.17 Thus it was proposed that an inappropriate intramedullary release of PDGF from megakaryocytes and/or platelets may ex- aggeratedly stimulate fibroblasts and collagen production thus resulting in fibrosis of the bone marrow.18 The pos-

sibility of a leakage of the growth factor was further supported by the morphological abnormalities of megakaryocytes and platelets which have been documented recently.19 Based on this hypothesis, several groups have evaluated platelet PDGF activity in MPD, as measured by [3H]-thymidine uptake. An overall decrease of intraplatelet PDGF levels in the different MPD was observed with the exception of idiopathic myelofibrosis, where some groups have shown reduced platelet PDGF activity, while others have reported increased platelet PDGF concentrations (for review, see Ref. 20). The discrepancy between these latter findings did not rule out the hypothesis of a leakagehelease of the growth factor from megakaryocytes and platelets, but it rather raised the possibility of an increased PDGF synthesis, at least in some cases.

Whatsoever, from these different studies it appeared that intraplatelet PDGF levels do not strictly correlate with the degree of fibrosis. Therefore, the release of PDGF, while undoubtedly inducing fibroblast proliferation, can- not account totally for the complexity of the myelofibrotic stroma and it seems probable that additional growth factors must be involved; the most important of which is likely to be transforming growth factor-0 (TGF-P). Actually, though PDGF displays a growth stimulatory activity on bone marrow fibroblasts greater than TGF-P, this latter factor appears to be the most powerful of the growth factors arising in platelets, in promoting the synthesis of collagen and fibronectin;Zl moreover, TGF-0 has been shown to regulate the mitogenic activity of PDGF and EGF on human marrow fibroblasts.22

lkansforming growth factor-fl (TGF-B): basic biology

TGF-P is a multifunctional cytokine capable of eliciting a wide variety of biological responses, depending on the target cell and growth ~onditions.23~2~ The mammalian TGF-P family consists of three isoforms, TGF-Pl, 2 and 3, which share most of their biological properties.25 TGF- fi is secreted in an inactive (latent) form which requires activation before it can exert its biological effects.26In their active form, all these three TGF-P isoforms are dimers of a 12 kDa peptide that arises from a larger precursor molecule.

Like PDGF, TGF-P is synthesized in megakaryocytes, stored in high concentrations in the alpha-granules of platelets and released at sites of injury.27 The TGF-P ef- fect most relevant to myelofibrosis is its potent regulatory activity on genes involved in the extracellular matrix synthesis. Actually, in idiopathic myelofibrosis, the myelofibrotic stroma is characterized by an increase in total collagen, that is interstitial and basement membrane col-

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TGF-P, MEGAKARYOCYTES & MPD 41

lagens, including type I, 111, IV and V.28 Moreover, increased deposition of adhesive glycoproteins, 1amini1-1,~~ fibronectin,3" tenascin3' and vitronectin3* has been documented in the advanced stages of the disease.

In this respect, TGF-f3 increases the biosynthesis of type I, 111 and IV collagens, fibronectin,33 as well as tenascin and proteoglycans.34 It simultaneously blocks matrix degradation by inhibiting the expression of collagenase- like proteases35 while enhancing the expression of pro- tease inhibitors, such as plasminogen activator inhibitor- 1.36 Thus both increased production and de- creased removal are equally likely to contribute to the ac- cumulation of pathological matrix in the disease. In addition, TGF-P modulates integrins expression in a man- ner that increases cell adhesion to the matrix.

Also, TGF-P has powerful angiogenic properties as demonstrated, when injected subcutaneously in newborn mice, with the occurence of new capillary formation at the site of injection;3' and an augmented growth of capillary blood vessels in the bone marrow is a well recognized fea- ture of idiopathic myelofibr~sis.~s A number of experi- mental studies underscore the potential role of TGF-P in fibr0sis.3~ In addition, there is strong correlative evidence to suggest that its overproduction is a problem in human diseases such as proliferative vitroretinopathy,@ liver cir- rhosis,41 pulmonary fibrosis42 or various fibrotic skin diseases including systemic scler0sis,4~ for example. In contrast, data concerning the possible implication of TGF- P in the development of myelofibrosis associated with haematological diseases is still sparce.

Megakaryocytes and TGF-p in myelofibrosis promotion

Significant findings were first reported in acute megakaryoblastic leukemia with associated myelofibrosis, where megakaryoblasts were shown to produce and secrete an active form of TGF-P; moreover media conditioned from megakaryoblasts were able to stimulate collagen synthesis by myelofibroblasts, the levels of collagen synthesis activity being suppressed, dose-dependently, by a specific anti-TGF-P antibody.44 Also, increased TGF-P plasma concentrations that correlate with increased bone marrow stromal turnover were further reported in acute micromegakaryocytic leukemia.45 Moreover, in a case report of acute megakaryoblastic leukemia manifesting liver tumor formation, immunolocalization of TGF-P was demonstrated in the leukemic cells of the bone marrow and the hepatic tumor. Further, the deposition of fibronectin and collagens I and IV was recognized in the extracellular matrix of both bone marrow and hepatic tumor, these results suggesting that specific expression of

growth factor proteins by leukemic cells may regulate the fibrosis of bone marrow as well as the tumor f0rmation.~6

Besides, results relevant to idiopathic myelofibrosis outlined the role that TGF-P is hypothesized to play in the medullary process. Actually, significantly increased. intraplatelet TGF-f3 levels were found in patients with agnogenic myeloid metaplasia compared to healthy subjects4' or patients with essential thrombocythaemia, which is an- other myeloproliferative disorder featured by expansion of the megakaryocytic ~ompartment.~* Further observations suggested that the increase of TGF-P platelet con- tent is reversible inasmuch as, in some patients, TGF-fi values returned to normal levels following IFN-y therapy.49

Recently, increased expression of TGF-P transcript andor elevated levels of the secreted TGF-P peptide measured in the conditioned media by patients peripheral blood mononuclear cells (PBMC) were reported.50 Moreover, using neutralizing antibodies to TGF-f3, the in- vestigators have shown that the TGF-f3 isoform predomi- nantly produced was the f3l isotype. According to this latter report, it appeared that, in idiopathic myelofibrosis, the regulation of TGF-P expression takes place at the transcriptional and post transcriptional levels, as suggested by the lack of regular correlation between the increase of the RNA expression and the production of the peptide.

Several types of cells are capable of producing TGF-P, including activated lymphocytes and monocytes/macro- phages. Interestingly, in PBMC of the patients with high levels of secreted TGF-f3 presented in the latter study, the peptide was localized in morphologically heterogenous cells characterized as cells of the megakaryocytic lineage at different stages of differentiodmaturation (Fig. 1). These results corroborate the demonstration of an increased number of megakaryocytes and their precursors in the peripheral blood of patients with agnogenic myeloid metaplasia, and further stress the probable link between TGF-P and megakaryocytes in the pathophysiology of myelofibrosis. The finding of normal TGF-P levels in essential thrombocythaemia, a myeloproliferative disorder where the megakaryocytes/platelets population is ex- panded, with increased mature megakaryocytes and elevated abnormal circulating platelets while myelofibrosis is not a common finding in this disease, strongly empha- sizes the crucial role of this growth factor in idiopathic myelofibrosis through its release from megakaryocytes. Undoubtedly, the megakaryocytic lineage is of major im- portance in the development of myelofibrosis, and the report of a steadily increasing percentage of myelofibrosis correlating with the proliferation of megakaryocytes in CML patients, pertinently underlines the correlation of this cell lineage to fibrosis.2

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42 M. MARTYRE

Figure 1 Co-localization of TGF-fl and gpII~II , , a megakaryocytic cell marker, in the peripheral blood mononuclear cells from a patient with idiopathic myelofibrosis. (A) Staining with a polyclonal TGF-PI antibody and subsequently with a TRITC-labelled secondary antibody. (B) Staining with a gpIIJII, monoclonal antibody and subsequent labelling with a FITC-goat anti-mouse antibody (original magnification X 400). (See Color Plate IV at the back of this issue.)

CONCLUSIONS

Megakaryocytes/platelets are the main source of TGF-P in humans; the role of the peptide in promoting increased expression of extracellular matrix molecules has been well documented. Besides, it is known that TGF-p can be sequestrated by binding proteins in the extracellular matrix, this process resulting in control of the avaibility of TGF- p, that is in regulation of its activity. TGF-p is inactivated by some of these proteins, such as a2-macroglobulin or decorin; in contrast thrombospondin-associated TGF-p is biologically active, so that this protein may act as a car- rier for TGF-p, favouring the deposition of active TGF-p in the tissue.5' Type IV collagen, fibronectin and soluble TGF-P type Ill receptor (betaglycan) may also act as reser- voirs for TGF-p, restricting its activity until it is released. Interestingly, type IV collagen deposition is augmented by TGF-P; fibronectin is known to bind to GPIlbAIIa (CD4la), one of the p3 integrin subgroup which is specif- ically expressed on cells of the megakaryocytic lineage, so that the interaction of the CD4la molecule with the de- posited fibronectin may participate to the mechanism(s) of fibrosis progression; finally, the type 111 receptor is a membrane-anchored proteoglycan, widely distributed52 it also exists in soluble forms that are released by cells and found in extracellular matrix; this receptor does not directly mediate the biological activities of TGF-p but may facilitate its interaction with the signaling receptor complex,-typeUtypeII receptors;53 moreover, the heparan sul- fate chains of betaglycan can bind basic fibroblast growth factor (bFGF), which is also present in megakaryocytic cells;54 bFGF has been described as a potent mitogen for

stromal fibroblasts and a potent inducer of angiogenesis, and it also appears to be involved in megakaryocytopoiesis.55

Excessive or sustained production of TGF-P thus appears likely to be a key molecular mediator of progressive deposition of extracellular matrix components and marrow fibrosis. Also, the prolonged release of sequestrated TGF-P subsequent to possible aberrations in the complex enzymatic machinery that regulates the activation of latent TGF-p, may result in locally raised TGF-p concen- tration, adding to the complexity of the fibrotic process. Finally, it must be reminded that TGF-p induces its own production by cells and this autoinduction may have a role in chronic progression of fibrosis, as well as the paracnne regulation of other potent cytokines, such as interleukin- 1 and tumor necrosis factor, which are also involved in tissue remodelling.

The recent cloning of thrombopoietin56 and its receptor,57 should by providing insights into megakaryocyte bi- ol0gy,5* facilitate the understanding of the mechanisms resulting in the excessive proliferation of megakaryocytes in myeloproliferative disorders.

In conclusion, megakaryocytes and TGF-P appear to play a major role in the development of myelofibrosis; however other cell lineages and pathogenic mechanisms need to be probed. Given the heterogenity of myeloproliferative disorders it seems likely that various pathways with complex hierarchical and interactive nature may con- verge to accomplish this process of fibrosis.

Acknowledgement pert secretarial assistance.

I am indebted to Ms. Agnts Birot for ex-

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TGF-P, MEGAKARYOCYTES & MPD 43

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tgf-β and megakaryocytes in the pathogenesis of myelofibrosis in myeloproliferative disorders

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