CD44 and Hyaluronan Binding by Human Myeloid Cells

Leukemia and Lymphoma, Vo . 2 I , pp. 407-420 Reprints available directly from the publisher Photocopying permitted by liccnse only

0 1996 OPA (Overseas Publishers Association) Amsterdam B.V. Published in The Netherlands by Harwocd Academic Publishers GmbH Printed in Singapore

CD44 and Hyaluronan Binding by Human Myeloid Cells

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FLORENCE SMADJA-JOFFE,’ STEPHANE LEGRAS,’ NICOLE GIRARD,* YUE LI,’ BERTRAND DELPECH,* FLORENCE BLOGET,3 KOHJI MORIMOT0,4 CAROLINE LE BOUSSE-KERDILES,’ DENIS CLAY,’ CLAUDE JASMIN’ and JEAN-PIERRE LEVESQUE5 ‘United’oncogtnhse Appliqute, Inserm U268, Hopital Paul Brousse, Villejuif, France, 2Laboratoired’Oncologie Molkculaire, Centre Henri Becquerel, Ilouen, France, 3Serviced’Anatomie et de Cytologie Pathologiques, Hopital Albert Calmette, 59037 Lille Cedex, 4Nihon University, School of Medicine Oyaguchi. Itabashi, Tokyo, Japan, 5Laboratoirede Biologie Cellulaire et Moltculaire des Facteurs de Croissance, Villejuif, France (Received July 27, 1995)

The CD44 cell surface molecule has been shown to be the principal cell surface receptor for hyaluronan (or hyaluronic acid), a glycosaminoglycan component of marrow extracellular matrix. However, its affinity for hyaluronan is not constitutive, since it depends on the cell type, the stage of differentiation and on activation by external stimuli including certain anti-CD44 antibodies and phorbol esters. Except for a few lymphoid cell lines, hematopoietic cells do not spontaneously bind hyaluronan and initial studies reported that, contrary to lymphocytes, rnyeloid cells could not be activated to bind hyaluronan. Because CD44 plays an important role in the initial phases of hematopoiesis, as shown by experiments using blocking anti-CD44 monoclonal antibodies, its capacity to mediate adhesion of primitive myeloid cells has been investigated. It was found that CD44 could mediate spontaneous adhesion to hyalurorian of immature myeloid cell lines KGI, KGla, and TFI, which serve as a model for hematopoit:tic progenitors. However, despite expressing high amounts of CD44, no more than 15% of bone marrow progenitors could adhere to hyaluronan. Recent experiments have shown that a very important feature of CD44 is its capacity to be rapidly activated by certain antibodies and cytokines (GM-CSF and KL) from a low affinity to a high affinity state for hyaluronan. These data shed light on striking similarities in the functional regulation of CD44 and of the two integrin receptors VLA4 (a4bl), and VLA-5 (a5bl), which are also expressed on hematopoietic progenitors. The relevance of these dai:a to the regulation of normal hematopoiesis and mobilization of CD34+ progenitors in the view of cell grafting is analyzed. In addition, we show that in idiopathic myelofibrosis, the amount of hyaluronan is markedly increased in the extracellular matrix from the myeloproliferative spleen. Considering that the production of cytokines is enhanced in this disease, we discuss whether CD44hyaluronan interaction may have a role in the pathophysiology of this myeloproliferative syndrome.

KEY WORDS: CD44 hematopoietic progenitors

integrins cytokines.

hyaluronan

adhesion

is constituted by an extracellular matrix, a repertoire of cytokines and cellular elements such as fibroblast-like cells, endothelial cells and adipocytes.1-5 Hemopoietic progenitor cells (HPC) are nested in this stroma where their proliferation and differentiation to the different hemopoietic lineages are hierarchically controlled by cytokines and ad-

INTRODUCTION

In adults, normal hemopoiesis takes place in the bone marrow, where hematopoietic progenitors are in close contact with the stroma, a specific microenvironment. This stroma Address for correspondence: Dr Florence Smadja-Joffe PhD, Director of Research, Unite dOncogenese Appliqute, Inserm U268. Hopital Paul Brousse, 14. Ave. P.V. Couturier, 94800 Villejuif Cedex France. Fax: 1-49-58-10-85.Tei: 1-39-58-10-67.

hesive interactions.f-s Adhesive interactions are critical for both homing of HPC to the bone-marrows-l’ and for longterm maintenance of their pluripotency Indeed, the dis-

*

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ruption of adhesive interactions by specific antibodiesprovokes the release of HPC into peripheral bloodI2-13and inhibits in vitro hemopoiesis,~2~~4 while in vitro cultures of HPC in the absence of bone-marrow stroma do not allow long-term self-renewal of immature pluripotent HPC. 15-17 Adhesive interactions are mediated by specific receptors which can bind to extracellular matrix components or to stromal cell counter-receptors. Collectively designated as cell adhesion molecules (CAMs), these receptors belong to different protein superfamilies such as integrins, selectins, immunoglobulin-likeproteins and CD44.I8-22Until now, functional studies on CAMs have mainly concerned cells of connectivetissues andof the immune system. These studies have shown functional similarities between integrins and CD44, particularly their association with cytoskeleton and their capacity to transduce intracellular signals when they are engaged with their specific ligands or with activatingmonoclonal antibodies.18.22-28Moreover, a very important common feature is their capacity to be rapidly activated by certain antibodies and cytokines from a low affinity state to a high affinity state for their ligand~.~9-” The CD44 molecule is strongly expressed on HPC,32-34and may play an important role in hemopoiesis since anti-CD44 monoclonal antibodies fully abrogate in vitro long-term hemopoiesis on preestablished strorna.14.34 However,the precise function of CD44 in hemopoiesis still remains largely unknown. It has been reported that CD44

is the principal cell surface receptor for hyaluronan (or hyaluronic acid),35 a major extracellular matrix component.36 Hyaluronan is a polycarboxylic glycosaminoglycan with a high molecular mass (>106) (Figure l).36 In the bone-marrow, it is synthesized in large amounts and secreted by stromal ~ells,3~-39 and accumulates in intercellular spaces (Figure 2). Biochemical studies show that hyaluronan is also associated with the fibroblastic cell surface.37-38 However, only few CD44 expressing cells, namely alveolarmacrophages,fibroblasts and certain lymphoid cells, spontaneously bind hyal~ronan.29,~~2 On the other hand, myeloid cells from bone marrow and various myeloid cell lines do not display significant affinity for hyaluronan.29.42Nevertheless, we have recently shown that very immature myeloid cell lines coexpressing in particular the CD34 antigen,43 were capable of binding hyaluronan significantly by means of CD44.U These results suggested that CD44 may be involved in the interaction of CD34’ HPC with hyaluronan. The aim of this review is to summarize data on the hyaluronan-binding function of CD44 in myeloid cells. The functional regulation of CD44 and its striking parallelism with the functional regulation of integrins a $ I and a=$Iwill be presented in the light of recently published data. The potential consequences of their dysregulation in the pathophysiology of idiopathic myelofibrosis will be discussed.

The hyaluronan molecule

OH

I

NH

‘CH3

D-glucuronic

acid

N-acetyl D-glucosamine

I

I

disaccharide unit Figure 1 Schematic structure of the hyaluronan molecule. Hyaluronan is a linear polymer of repeating disaccharidic units constitued of D-glucuronic acid (1-8-3) N-acetyl-D-glucosamine ( I -8-4). Its molecular mass can reach several millions.

82

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60P

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F. SMADJA-JOFFE ETAL.

THE CD44 MOLECULE

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Structure CD44 is a transmembrane cell surface glycoprotein with multiple isoforms which is present on a range of different cell types including hematopoietic cells. It is encoded by a single gene of approximately 60kb located on chromosome 11~1345and comprising at least 20 exons.M.47 The hematopoietic CD44 isoform, also known as the standard form of the molecule (CD44s) is encoded by ten exons (designated exons 1s to 10s). It is synthesized as a 37kD molecule that is processed to a 80-9OkD molecule by glycosylation on its extracellular domain with N- and 0-linked oligosaccharides as well as by chondroitin sulfate chains (Figure 3).48-51 There is usually no (or only a very low) expression of the other ten exons. However, in various neoplastic cells and after cell activation (reviewedby Gunthert et UP), they generate multiple isoforms by alternative splicing of the mRNA in differentcombinations.These isoforms can comprise up to 420 additional amino acids inserted extracellularly, and further glycosylation of the inserted peptidic sequences can yield to variant molecules with molecular mass higher than 200kD. The inserted regions are very hydrophilic and therefore susceptible to display additional binding features.52.53 The CD44 molecule is bound to the actin-based cytoskeleton,by the means of an ankyrin-like protein, a membrane-associated cytoskeletal protein, and of the ERM (Ezrin, radixin and moesin) family proteins localised just beneath the plasma membrane;5’58 it is also associated with the protein kinase C.59 Phosphorylationof the intracytoplasmicdomain of the molecule (on at least two serine residues, Ser323 and Ser325) is reversible and might play a role in the regulation of the CD44-cytoskeleton interaction>- However, this role is still unclear, and data remain contradictory,perhaps because mechanisms are different according to the cell type. Thus, it was reported that in lymphoma cells CD44 binding to ankyrin-derivatized beads was enhanced by its p h o s p h o r y l a t i ~ nOn . ~ ~the ~ ~other ~ hand only nonphosphorylated CD44 molecules were linked to the cytoskeleton61 in peritoneal macrophages and, in epithelial cells and fibroblasts, phosphorylation was not required for CD44 binding to the cytoskeleton.54.56.64.65

gions, that comprises a particularly important arginine residue at position 41, is situated near the NHz terminus and is homologous to other hyaluronan-binding proteins. The other region is more membrane proximal and lies outside the hyaluronan-bindingprotein homologous domain. The presence in the variant isoforms of CD44 of additional peptidic sequences which are very hydrophilic and highly glycosylated, is susceptible to interfer in the CD44 hyaluronan interaction. It has been reported that the isoform CD44-Rl, which is characterized by a 132 amino acids insert coded by exons v8 to v10 has no affinity for hyaluronan.68 However, it is likely that the altered functional activity of this isoform was not due to the insert but merely to mutation of one to 3 amino acid residues.@ Transfection experiments with deletion mutants have demonstrated that the cytoplasmic domain of CD44, and in particular the last 52 amino acids, was also critical for binding of hyaluronan to the CD44 extracellular domain.70.7’ Indeed, it has been shown that phosphorylation of the cytoplasmic domain might have a role in regulation of the CD44-cytoskeleton interaction,59-70and this interaction is likely to control the affinity of CD44 for hyaluronan by changing the conformation of the molecule and its distribution on cell s~rface.29,[email protected] This “inside-out’’ regulatory pathway may display some variations in distinct cell types, thus accounting for the role of the cellular environment in the functional regulation of CD44.73 Indeed, many CD44-expressingcells do not bind hyaluronan, and transfection of identicalCD44 constructs into different cell types conferred the hyaluronan-binding phenotype only in certain cell types.73 Regardinghematopoieticcells, studies have shown that lymphoid cells from peripheralblood, spleen or bone-marrow did not spontaneously bind hyaluronan, but it was possible to induce hyaluronan-bindingby various stimuli such as phorbol ester, certain monoclonal antibodies and cytokines.29.42.70-75 It was first reported that unlike lymphocytes, CD44 expressing myeloid cells were unable to bind hyaluronan, even after treatment with phorbol ester and certain monoclonal antibodie~.29-~2 In the following sections of this review we will expose recent data showing that primitive myeloid cells, in particular CD34+HPC,can bind hyaluronan, and that this binding can be activated by external stimuli including cytokines.

Hyaluronan binding Binding of hyaluronan by CD44 depends upon an ionic interaction between negatively charged carboxyl groups of the glycosaminoglycan and two regions from CD44 extracellular domain, containing clusters of positively charged basic amino a~ids.66.6~ Using truncation and sitederived mutagenesis, it was shown that one of these re-

CD44 EXPRESSION ON NORMAL MYELOID CELLS Expression of CD44 on hematopoietic cells has been mostly studied using monoclonal antibodies that recognize indistinctly standard and variant CD44 molecules.

CD44 AND HYALURONAN BINDING B Y MYELOID CELLS

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Extra&uIaldanain # 250 amino acids coded by emns 1s to 7s

Transmesnbnmedomain # 20 amino acids coded by exon 8s

I

Intracytc~dcdomain # 70 amino acids co&dby exolls A and 10 s

CObH

LEGENDS

Figure 3 Schematic structure for the hematopoietic (standard) form of the CD44 molecule. The hematopoietic (standard) form of the CD44 molecule is encoded by exons 1s to 10s. Hyaluronan binding is cooperative between two basic regions in the extracellular domain. One is near the NH2 terminus and is homologous to other hyaluronan binding proteins; in this region, Arg4l has a critical role in hyaluronan binding. The other region is outside the hyaluronan binding Pornologous domain. The last 52 amino acids of the cytoplasmic domain play also a role in hyaluronan binding to the extracytoplasmic domain. (See Color Plate XVIII at the back of this issue.)

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These studies have shown that the amount of CD44 antigen varies according to the lineage and the stage of maturation. Thus, it is high on CD34+ hematopoietic progenitor cells (HPC) and mature granulocytes, but down-modulated during the intermediate stages of maturation.32-34.76 On the other hand CD44 expression remains high through differentiation into monocytes and macrophages.77 In addition, within the population of CD34+HPC, a high heterogeneity in CD44 expression is observed,33.34,unpublisheddataand it is not yet known whether this heterogeneity is also related to the stage of maturation, like CD34, CD38 and HLA-DR antigens.78.79Indeed, CD34' HPC comprise a continuum of cells at different stages of maturation. The most primitive HPC are multipotent, capable of self-renewal providing they are seeded onto a preestablished str0ma16.1~ and with a high proliferative potential. On the other hand, the most mature HPC are endowed with a low proliferative potential and their differentiation is restricted to a single cell lineage.78.79 Interestingly, the most primitive HPC designated as "long term culture initiating cells" (LTCIC), strongly express CD44.34 Moreover, antibodies to CD44 can abrogate their growth, whereas they were ineffective on the development of more mature CD34+HPC.34 Taken together, these data strongly emphasize the important role of CD44 at an early stage of hemopoiesis. Unlike in normal intermediate myeloid cells, there is no down-regulation of CD44 in blasts from patients with acute myeloid leukemia. A study on about 40 leukemic patients reported that CD44 expression on these cells was as high as on CD34+ HPC. (Legras et aZ., manuscript in preparation). This impairment of the CD44 down-regulation process may only be a consequence of the blockage of myeloid differentiation in leukemia. However, it cannot be excluded that it may also have a role in this blockage itself and it may be relevant to investigate this hypothesis further. Established myeloid cell lines express different levels of CD44. As shown in Table 1, CD44 expression is particularly high on the most immature of them, i.e. KG1, KGI a, TF1 and M07 cells, whose differentiation has been "frozen" at an early stage of maturation, as assessed by coexpression of the CD34 antigen.42.77.8&82Moreover, the growth of some of them (TF1, M07) is cytokine dependent as in the case of HPC. Therefore, these cell lines might be interesting models for studying the function of CD44 in HPC. In contrast, CD44 expression is low or undetectable on cells which are more mature and CD34 negative, such as HL60 HEL, K562 and UT7 cells.83-86 (unpublished results and Table 1). The U937 cell line, which is CD44highand CD34"'g may represent an exception since it derives from a patient with monoblastic

leukemia and CD44 is not downregulated during monocytic differentiation.74Finally, several variant isoforms of CD44 are lightly expressed by normal myeloid cells. Thus, a faint protein expression of variant exons v8 to v10 has been detected in a subset of HPC by cytofluorometry analysis.87 Exons v3 and v6 were also slightly transcribed, as shown by RT-PCR analysis (Reverse transcriptase polymerase chain reaction) but no protein expression could be detected by cytofluorometry. On monocytes, a slight protein expression of the variant exon v3, v6 and v9 was detected by cytofluorometry.88.89 and ""Published results

REGULATION OF MYELOID CELL ADHESION TO HYALURONAN Bone-marrow cells, despite expressing CD44, do not bind hyaluronan;25s42since hyaluronidase treatment does not result in hyaluronan binding, this binding inability is due to the low affinity of CD44 for this ligand, and not to a masking of the binding site by endogenous hyaluronan.42 Moreover, it has been shown that neither bone-marrow cells nor established myeloid cell lines [murine: M 1.16, Wehi 3, 745.6, and DIB.10 or human: U937, K562 and HL60] could be activated to bind hyal~ronan.~9,~~,~.73,~~ However, recent data strongly suggested that the hyaluronan binding function of CD44 might be regulated according to the stage of myeloid differentiation. Indeed, it was first reported by Morimoto et aZ.43 that the myeloid cell lines KG1 and KGla spontaneously adhered to hyaluronan via CD44, and the same was observed in the case of TFl and M07 cells (Table 1 and manuscript in preparation). Since these cell lines are clonal populations of very primitive myeloid cells that, like hematopoietic progenitors, express the CD34 antigen,42 these results suggested that CD34' hematopoietic progenitors from the bone marrow might also adhere to hyaluronan. Hematopoietic progenitors could not be detected in earlier experiments because of their very low proportion (less than 0.1 %) in the normal bone marrow.16 Therefore, using standard purification procedures we prepared bone marrow cell populations comprising more than 95% of CD34+ cells, and analyzed their hyaluronan binding ability. We found that about 15% of CD34+ cells spontaneously adhered to immobilized hyaluronan. CD44 expression on adherent cells was brighter than on the non-adherent ones, indicating that, as otherwise reported in the case of lymphocytes and KG1 cell~,29~*.74 a high amount of CD44 was necessary for adhesion. However, only a small proportion of CDMhighprogenitors adhered to hyaluronan, indicating that these cells express both low- and high-affinity molecules.

f W

-/low

+

+++

+++ +++

+++ +++

low No

+/-

+

+

NO

No

No

NO

No

No

'I

No

NO

9

++(SO%)

++ (13%)

+++ (70%)

++(> 80%)

++ (20%)

Megakaryoblastic Leukemia

-

+ -/flow

+

-

Yes Yes

No

UT7

-

+

-

-

low

+++

low

No

u937

HL60

KS62

TF I M07

KGla

+

++(> 80%)

Erythroleukemia

++ (40%)

HEL

No

Slirnulutiun

++

-

+

Spontuneuu.\

++

C D I4

C D i'5

-

+

+++

+++

HLA-DK

Hyaluronan binding

+/-

KG I

CD38

CD34

CD44

Antigenic features

Cytokinedependenr growth

Acute Myeloblastic Leukemia Acute Myeloblastic Leukemia Erythroleukemia Megakaryoblastic Leukemia Chronic Myeloid Leukemia Promyelocytin Leukemia Monocytic Leukemia

Origin

Cell line

Table 1 CDU-mediated hyaluronan binding by human myeloid cell lines

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F. SMADJA-JOFFE ETAL.

Interestingly, an antLCD44 monoclonal antibody, designated H90,23 could rapidly increase adhesion of CD34+ progenitors to hyaluronan, without an increase in CD44 expression, showing that CD44 molecules in a low-affinity conformationcan be converted into a high-affinity conformation (Legras et al., manuscript in preparation). This also suggested that adhesion of progenitors to hyaluronan might be activated in vivo by physiological stimuli which, by analogy with recent dataon %PI anda& integrins,30*3l might be cytokines. This hypothesis, currently being investigated in our laboratory, is supported by preliminary results showing that the granulocytic-macrophagestimulating factor (GM-CSF), IL3 and kit ligand (KL), increase adhesion of TF1 cells to hyaluronan (Figure 4). In addition, experiments carried out on KGl and KGla myelojd cell lines have shown that CD44 can also be activated by phorbol e~ter.4~ This alternativeactivation pathway is slow (about 16 hours) and requires new protein synthesis. A similar mechanism for CD44 activation has also been observed in lymphoid cells.70J2 Nevertheless, considering the very strong expression of CD44 on HPC, the proportion of hyaluronan adherent HPC remains surprisinglylow, even after activation (about 50%).Further research will eventually determine whether cytokines could synergize to increase the proportion of hyaluronan-adherentHPC. Alternatively, it has been oth-

500 c.,

1 400

I --

2

.d

ccr

0

$ 300

-

erwise shown that CD44 might have affinity for extracellular matrix molecules other than hyaluronan, namely for fibronectin, chondroitin sulfate and collagen.65.9'J.91In addition, Verfaillieef al.92 showed that CD44 did not directly mediate adhesion of committed human HPC to fibronectin, but cooperated with the integrin a 4 P I for this adhesion. We did not detect progenitor adhesion to chondroitin sulfate, and whether CD44 is involved in HPC adhesion to collagen has not been yet studied. Serglycin, an hematopoietic specific proteoglycan, has been recently described as a new ligand of CD44.93For this reason, we are currently investigating whether CD44 could mediate adhesion of CD34' HPC to serglycin. While in the bone marrow cell population only CD34+ hematopoieticprogenitors adhere to hyaluronan, it has recently been reported that some mature myeloid cells present in the peripheral blood can also bind hyaluronan. Thus, platelets can bind hyaluronan by the means of CD44;94 peripheral blood monocytes also bind hyaluronan after having matured in Interestingly, the appearance of their hyaluronan-binding function is associated with expression of multiple large molecular weight isoforms, showing that in these cells CD44 function and expression are both regulated in a differentiation-dependent way. It has been reported that hyaluronan stimulates granulocytic function;95 however, the involvement of

T

T

GM-CSFIHA

KUHA

--

mi 0 0

c . '

c

200

--

100

--

E

4

0-

BSA

NIUHA

Figure 4 Stimulation of CD44-mediated adhesion to hyaluronan by the cytokines GM-CSF and KL. TFI cells labelled with W r and incubated in

RPMI 1640 medium plus 0.2%BSA, were seeded onto hyaluronan-coated microwells (/HA) for 15 mn at 37°C in the presence of lOng/ml of indicated cytokines. After removing the non-adherent cells by three washes with the same medium, adherent cells were lysed with SDS:NaOH and their radioactivity was measured. 100%is the radioactivity of spontaneously adherent cells. NIL: no added cytokines; BSA: microwells coated with bovine serum albumin.

CD44 AND HYALURONAN BINDING B Y MYELOID CELLS

CD44 in this interaction has not been demonstrated and we have not as yet detected adhesion of granulocytes on immobilized hyaluronan (unpublished results).

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COMPARAISON OF THE FUNCTIONAL REGULATION OF CD44 AND INTEGRINS VLA-4 AND V L A J The affinities of CD44 and of the two integrin receptors VLA-4 (cc4pI, CD49cVCD29) and VLA-5 (cc5PI, CD49eKD29) for their respective ligands are both regulated by mechanisms involving protein k i n a ~ e s59. ~96~ However, unlike PI integrins, which are constitutively phosphorylated,31 CD44 phosphorylation seems to be an inducible and a reversible phenomenon,61.62 suggesting that the mechanisms regulating the affinity of these adhesion molecules might not be strictly identical. However, striking similarities could be underlined. CD44, VLA-4 and VLA5 are strongly expressed on CD34' HPC with a low affinity for their ligands, since no more than 15% CD34' HPC spontaneously adhere on hyaluronan and only about 5% of them adhere on fibronectin 1831-34 97398 and era[. I n p w a a t l o n ) Like with CD44, blocking of VLA6 with anti-VLAA-4 and anti-CD29 antibodies fully abrogates lymphopoiesis arid drastically inhibits myelopoiesis.1*.14 It has been recently shown that these two integrin receptors can be transiently activated by mitogenic cytokines such as interleukin-3 (IL3), granulocytemonocyte colony stimulating factor (GM-CSF) and KIT ligand (KL).31 Furthermore, combinations of these cytokines confer more than 50% of HPC with an adherent phenotype on fibronectin (Levesque et al., manuscript in preparation). Among the p l integrins expressed by HPC, this activation is specific for VLA-4 and VLA-5 since adhesion on collagens, laminin and vitronectin is not augmented. Our preliminaq experiments show that in the model cell line TF1,81 CD44-mediated adhesiveness on hyaluronan is also transiiently augmented by these cytokines (Figure 4).

CD44-HYALURONAN INTERACTION IN THE PATHOPHYSIOLOGY OF THE IDIOPATHIC MYELOFIBROSIS Idiopathic myelofibrosis, or myeloid splenomegaly, is a chronic neoplastic disorder characterized by bone-marrow fibrosis, extramedullary hemopoiesis, splenomegaly and a leucoerythroblastic blood picture.99 Using G6PD isoenzyme analysis, it has been demonstrated that cells from the three myeloid lineages belonged to the same patho-

415

logical clone.IM) In this disease, circulating HPC, including pluripotent and lineage-restricted progenitor cells, are increased up to 160-fold that of control blood.lO1 and Lc Kerdil&era/ (manuscnpt i n preparation) s' ince splenectomized patients have decreased levels of circulating progenitors, it has been suggested that the spleen plays a major role in progenitor cell overproduction, release or storage.102 It is conceivable that the abnormal homing of circulating progenitors in the myeloproliferative spleen could be enhanced by the unusual deposition of hyaluronan (Figure 5 and"J3), to which hematopoietic progenitors might adhere by the means of CD44. As it mentioned above, this adhesion is likely to depend on activation by cytokine. It would therefore be of interest to see whether CD44 can be activated by TGFP, since the amount of this cytokine in the serum is strikingly increased during the course of this diseaselW and since this cytokine can act on hematopoietic progenitors.105 It would also be relevant to investigate in serum of these patients for the presence of cytokines such as GM-CSF, KL and IL3, whose stimulating effect on CD44 and VLA4 binding activity has already been established.31~ d ~ u b l = h e d ~ eIn ~ uaddition, lt~ a local production of these cytokines in the spleen, as reported in a murine experimental model of idiopathic myelofibrosis,1% may also contribute to the abnormal homing of hematopoietic progenitors in this organ.

CONCLUSION Adhesive interactions with bone marrow stroma are critical for homing of HPC in this organ, for the long-term maintenance of their pluripotency and most probably for regulating their differentiation into mature progeny. Therefore, it is of great importance for a comprehensive study of normal hematopoiesis to determine which moiecules are involved in these interactions and how their activity is regulated. CD44, like the p 1 integrins VLA-4 and VLA-5, plays an important role in hematopoiesis. Here we have reviewed data showing that CD44 can mediate adhesion of CD34' HPC to the hyaluronan component of bone-marrow extracellular matrix. This adhesion to hyaluronan might not only place HPC in close contact with the various regulators present on the fibroblastic cell surface, but also play a part in the functional regulation of HPC proliferation anddifferentiation, since hyaluronanCD44 interaction is capable of triggering intracellular signaling. Further studies should elucidate this process. In addition, a very important feature of CD44 is its ability, like the P I integrins VLA-4 and VLA-5, to be activated by certain antibodies and cytokines including GM-CSF, IL3 and KL. thereby switching from a low

416

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5

Figure 5A Hyaluronan staining in normal spleen and in spleen from patients with idiopathic myelofibrosis. Specific staining of hyaluronan, as well as the negative controls, were performed on histological sections prepared from frozen (5A) or formalin fixed (5B and 5C) samples, as described in legends for Figure 2.5A: N o m l spleen. The staining of hyaluronan was restricted to the adventitia of artery (5A). (See Color Plate XIX at the back of this issue.)

affinity to a high affinity state for hyaluronan. Furthermore, preliminary data revealed in this review suggested that this action of cytokines was transient and dose-dependent like in the case of VLA4 and VLA5.31 A thorough study of these regulatory mechanisms is likely to improve the use of cytokines for mobilizing HPC into the circulating blood, as shown with G-CSF,107-108 prior to autograft, and for increasing their seeding efficiency in the recipient bone-marrow.'08.1@ Finally, a dysregulation of the affinity of CD44 for hyaluronan together with the impairement of hyaluronan synthesis by stromal cells, may play a part in the pathophysiology of idiopathic myelofibrosis, i.e. in myelopoiesis which develops in the spleen, thereby contributing to the striking enlargement of this organ.

Indeed, it has been shown that GM-CSF is overproduced and that hyaluronan accumulated in the spleen extracellular matrix during the course of this disease. It is therefore tempting to suggest that under the action of GM-CSF, CD44 present on circulating HPC may be maintained under a high affinity conformation for hyaluronan, thereby enhancing adhesion of HPC to hyaluronan already accumulated in the spleen. This process may thus contribute, together with the impairment of other adhesion molecules such as VLA4 and VLA5, to the abnormal homing of HPC in the spleen and to the onset of ectopic myelopoiesis. If this model is confirmed, it may become possible to diminish ectopic myelopoiesis by modifying the CD44-hyaluronan interaction.

CD44 AND HYALURONAN BINDING BY MYELOID CELLS

417

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5B

5c

Figure 5B and 5C: Spleen from patients with idiopathic myelofibrosis. The staining of hyaluronan was strong and extensively distributed through extracellular matrix of the myeloproliferative spleen (5B). The staining was suppressed after Srrepromyces Hyaluronidase digestion (512). (See Color Plate XX at the back of this issue.)

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REFERENCES

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1. Trentin, J. J. (1970) Influence of hematopoietic organ stroma

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CD44 AND HYALURONAN BLNDING BY MYELOID CELLS

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