191

Biochimica et Biophysica Acta, 1032 (1990) 191-211 Elsevier

BBACAN 87229

Proteoglycans in haemopoietic cells S.O. Kolset 1,. and J.T. Gallagher 2 Institute of Medical Biology, University of Troms6, Troms6 (Norway) 2 and CancerResearch Campaign Department of Medical Ontology, ChristieHospital, Manchester (U.K.) (Received 18 July 1990)

Contents I.

Introduction - The haemopoietic system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

192

II.

Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Structural characteristics of proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Glycosaminoglycans of haemopoietic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Protein - glycosaminoglycan linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

192 192 193 193 195 196

III.

Exocytosis and degradation of proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

197

IV.

Protein cores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Proteoglycans in secretory granules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Invariant chain proteoglycan in lymphocytes and macrophages . . . . . . . . . . . . . . . . . . . . . . .

197 197 198

V.

Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199

VI.

Monocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

200

VII.

Leukaemic cells in the myeloid series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

201

VIII. Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

202

IX.

Mast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mast cell heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Glycosaminoglycan components of mast cell proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Mucosal mast cells (M-MC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Connective tissue mast cells (CT-MC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Human mast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Proteoglycans and mast cell development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Functions of mast cell proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

202 202 203 203 203 204 204 205

X.

Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. T-lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. B-lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

206 206 206

XI.

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

208

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

208

* Present address: La Jolla Cancer Research Foundation 10901 North Torrey Pines Road La Jolla, CA 92037, U.S.A. Abbreviations: Ii, invariant chain; CS, chondroitin sulphate; HA, hyaluronic acid; CS-A, chondroitin-4-sulphate; PMN, polymorphonuclear granulocytes; PMA, phorbol 12-myristate 13-acetate; CSPG, chondroitin suphate proteoglycan; LDL, low density lipoprotein; M-MC, mucosal mast cells; CT-MC, connective tissue mast cells; NK, natural killer cells; GAG, glycosaminoglycans. Correspondence: J.T. Gallagher, Cancer Research Campaign Department of Medical Oncology, Christie Hospital, Manchester, M20 9BX, U.K. 0304-419X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

192 I. Introduction - The haemopoietic system

In humans and other mammals haemopoiesis takes place in the bone marrow and fetal liver. Due to the rapid turnover of cells in the peripheral blood, the human body must produce about 4.1011 cells per day [1]. This high demand for cell renewal is met by an extremely efficient mechanism for cell amplification in which a small number of pluripotent stem cells differentiate and proliferate to produce mature cells that are released into the blood circulation system. Basically, there exist two major pathways of development; the myeloid and lymphoid lineage. The myeloid progenitor cells are precursors for the granulocytes, monocytes, platelets and mast cells, and lymphoid progenitors develop into B- and T-lymphocytes, as illustrated in Fig. 1. The development and maturation of the various blood cell types is dependent upon a complex interplay between different cytokines [1,2] and the modulatory role of different microenvironments in the marrow stroma, which include cell surface and extracellular matrix proteoglycans [3,4]. The different blood cells perform highly diversified functions, ranging from the production of antibodies by B-lymphoyctes to the killing of bacteria by polymorphonuclear leukocytes and the participation of platelets in the coagulation process. All the cells contain proteoglycans which contribute significantly to the diverse functions of the haemopoietic system. The major haemopoietic cell proteoglycans have a distinctive molecular design which was first described

for the heparin proteoglycan (see section IX). Variations in structure of this type of proteoglycan are largely due to differences in the composition of the glycosaminoglycan chains, which in some instances represent markers of cellular phenotype. Other classes of proteoglycan are the glycanated forms of the invariant chain (Ii) and the Hermes lymphocyte homing antigens (see sections IV-B and X-B). Small quantities of heparan sulphate have also been found on the surfaces of some leukaemic cells (section VII) and the proteoglycan known as syndecan, which contains heparan sulphate chains has been detected on immature B cells and plasma cells (section VII). The purpose of the present paper is to review in further detail the biochemical properties of proteoglycans in cells belonging to the haemopoietic system, and to provide some perspective on the functional implications of these acidic macromolecules in the various cell populations. A short introduction will give a general outline of proteoglycan structure and some general features on the turnover of these molecules in haemopoietic cells. Thereafter a presentation of the protein cores of those proteoglycans that have been characterized in detail is presented, followed by a separate section dealing with the individual cell types. Finally, various structural and functional aspects of the different proteoglycans are discussed. II. Proteoglycans

H-A. General features

[/ON[[

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FUNCTIONS

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Lb,mphs!]n,~B LYI~PHOCY]FS ibg m(i 'T[I ~

Synthesis of antibodies Cell mediated immunity

rn!'c?~'T"t~

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Coagulation

GR~U[ OC'fTES Ei!si nophi l t, Ba ~41hi ! s ",cut, ,:ph i 1,,

inflammation Phagocytosis, microbial killing

',If)N(,I[ y TES 'HACROpIIAP~ES

Antigenpresentation Phagocytosis

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Inflammatory Responses, Coagulation Oxygen transporter

Fig. 1. A schematic outline of haemopoiesis. A small population of stem ceils in the bone marrow is believed to give rise to all lymphoid and myeloid cell types in the blood and tissues [1,11]. The functions outlined are limited to the major characteristics of each cell type. There is a considerable amplification in cell numbers as stem ceils differentiate and mature to functional 'end-cells'.

Proteoglycans are produced by all types of mammalian cells. By definition, they consist of one or more sulphated polysaccharide chains, the so-called glycosaminoglycans, covalently linked to protein. This simple definition encompasses a vast range of macromolecules that vary in molecular size, composition and function. Proteoglycans were once considered to be mainly structural components because they were originally identified in connective tissues such as cartilage and skin, where they occur in relatively high concentration in the extracellular matrix and play well-established roles in determining the physical properties of the tissues. However, with the development of sensitive methods of detection, including immunocytochemical techniques [4a] and the specific metabolic radiolabelling of cells in culture, it is now widely recognised that proteoglycans are not confined to mesenchymal tissues, nor indeed to the extraceUular matrix. For instance, they are important constituents of plasma membranes and basement membranes of epithelial, endothelial and neuronal cells, and they are present in storage granules of immunosecretory and neurosecretory cells [5,5a]. Glycos-

193 aminoglycans have even been detected in the cell nucleus. Other intracellular organdies such as lysosomes and endosomes also contain glycosaminoglycan components. This rich variety of anatomical locations and the well-documented diversity of proteoglycan structure clearly indicates that there is no single functional attribute of these macromolecules. Each proteoglycan species requires separate evaluation of its biological properties. An indication of the functional variability can be gained from consideration of cell surface proteoglycans. These proteoglycans influence the interactions between cells and their microenvironment mediating both direct cell/cell binding and adhesive interactions between cells and structural proteins of the extracellular matrix. One of the TGF-fl binding proteins at cell surfaces is a proteoglycan [6,7] and lipoprotein lipase binds to proteoglycans on adipocytes and endothelial cells [8,9]. Plasma membrane proteoglycans can also bind and 'present' growth factors (for reviews see Refs. 10 and 11). For example, stromal cells of bone marrow produce proteoglycans that bind haemopoietic cell growth factors and neuronal cell surface proteoglycans form complexes with mitogens that stimulate the proliferation of Schwann cells. Some plasma membrane proteoglycans may also have an intrinsic capability of regulating cell growth. Examples are the anti-proliferative activities of distinctive types of heparan sulphate produced by confluent smooth muscle cells [12] and hepatocytes [13]. In the latter case, the growth inhibition is correlated with the transfer of heparan sulphate from the cell surface to the nucleus.

H-B. Structural characteristics of proteoglycans Some well-characterised proteoglycans that give an indication of the wide range of molecular species in the proteoglycan family are illustrated in Fig. 2. The major proteoglycan in cartilage, designated as aggrecan, is a very large molecule containing over one hundred glycosaminoglycan chains of the chondroitin sulphate and keratan sulphate types linked to a 207 kDa core protein [13a]. The amino terminal domain of this protein binds with high affinity to hyaluronic acid and the aggregation of large numbers of proteoglycan monomers along a central hyaluronic acid 'filament' produces supramolecular aggregates that are responsible for the compressive resilience of the cartilage matrix. Much smaller proteoglycans are found in many connective tissues, including cartilage, skin and sclera. One of these consists of a small core protein (40 kDa) containing only one chondroitin or dermatan sulphate chain. This proteoglycan has recently been called 'Decorin' because it binds in a regular array to collagen fibrils [14]. Proteoglycans of plasma membranes often contain

heparan sulphate as the glycosaminoglycan. These proteoglycans are commonly intercalated in the hpid bilayer by means of a hydrophobic sequence of amino acids or by a covalently linked inositol-phospholid [15,15a]. A membrane proteoglycan initially isolated from mouse mammary epithelial cells (Fig. 2) has been named 'syndecan' (from the Greek 'syndein' meaning to 'bind together') because of its probable role in stabilising the epithelial layer through the binding of its heparan sulphate chains to the interstitial matrix [16]. One of the largest core proteins (400 kDa) is found in the heparan sulphate proteoglycan of basement membranes produced by the EHS sarcoma [17,18]. This proteoglycan plays an important role in stabilising the molecular architecture of the basement membrane through interactions with other structural components such as laminin and collagen type IV [19]. A similar or identical proteoglycan is present in the basement membranes and the interstitial matrix of many normal tissues [20,20a]. In the foregoing examples, the polysaccharide chains of the proteoglyans are often found in specific domains of the protein core separated by short sequences of proteinase-sensitive amino acids. However, in the highly characteristic secretory granule proteoglycan of mast cells, basophils and NK cells the polysaccharide chains, which may be heparin or chondroitin sulphate, are tightly packed along a relativly short peptide region of the core protein. This glycanated peptide, which is resistant to proteolytic degradation, has an unusual amino acid sequence which is present in only one other proteoglycan, the so-called L-2 proteoglycan produced by a rat yolk sac tumour [21]. Curiously, a closely related protein is encoded by the per gene which controls the circadian rhythms in Drosophila, and this protein is substituted with glycosaminoglycan chains [22,23].

H-C. The glycosaminoglycans The glycosaminoglycan components of proteoglycans are sulphated polymers composed of repeating disaccharide units (Table I). The chain length can vary from 25 to 150 disaccharides. With one exception, keratan sulphate, the disaccharide unit consists of an amino sugar and a uronic acid. The glycosaminoglycans are synthesized by membrane bound enzymes in the Golgi system which polymerise monosaccharide precursors onto protein core acceptors. Sulphation of individual sugars occurs at the polymer level, either concurrently with polymerisation or after polymer synthesis is complete (for reviews see Ref. 24 and 24a). Perhaps the most common glycosaminoglycan is chondroitin sulphate in which the disaccharide consists of N-acetylgalactosamine (GA1NAc) and glucuronic

194 acid (GlcUA), the amino sugar being sulphated at C-4 or C-6 to form the type A or type C disaccaride units: GalNAc-4 or 6S ,81 ~ 4 GlcUA

An isomeric form of this disaccharide is found in dermatan sulphate where the major disaccharide contains iduronic acid rather than glucuronic acid. Iduronate is normally linked to the 4-sulphated isomer of GalNAc:

tin s u l p h a t e will c o n t a i n b o t h t y p e A a n d t y p e C d i s a c c h a r i d e s , a n d d e r m a t a n s u l p h a t e is i n v a r i a b l y a copolymer of iduronate- and glucuronate-containing d i s a c c h a r i d e units. T h e s u l p h a t i o n p a t t e r n s o f t h e s e g l y c o s a m i n o g l y c a n s m a y b e f u r t h e r c o m p l i c a t e d b y the p r e s e n c e of d i s u l p h a t e d d i s a c c h a r i d e s . I n the t y p e E d i s a c c h a r i d e s o f c h o n d r o i t i n s u l p h a t e b o t h C-4 a n d C-6 o f t h e a m i n o s u g a r are s u l p h a t e d : GalNAc-4S,6S ,81 -* 4 GIcUA

GalNAc-4S ,81 ---,4 IdUA

In reality the chondroitin and dermatan sulphates often consist of mixed disaccharide isomers. Thus, chondroi-

whereas in dermatan sulphate an extra sulphate group may be present on C-2 of the iduronate component. These are called di-B-type disaccharides: GalNAc-4S ,81 ~ 4 IdUA-2S

A

Cartil age proteoglycan

G2

GI

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In rare instances, glucuronic acid is also sulphated at C-2. In heparin and the chemically related heparan sulphate, the amino sugar is glucosamine and the hexosaminidic linkage is in the a configuration. The glucosamine unit is N-sulphated (GlcNSO3) or N-acetylated (GlcNAc) and the hexuronate may be glucuronate or iduronate. In heparin, over 80% of the glucosamines are N-sulphated and the most common disaccharide [24] is a trisulphated derivative of structure: GlcNSO3-6S al ~ 4 IdUA-2S

Although present in only small quantities, N-acetylated disaccharides in heparin are very important for the biochemical properties of the polymer. They are components of the specific antithrombin binding sequence in

Hydrophobic FeglOrJ - -

-

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Fig. 2. Proteoglycan structures. Different types of mammalian proteoglycans are illustrated. (A) The aggregating proteoglycan of cartilage (aggrecan) is a large macromolecule in which the core protein (207 kDa) is substituted with over 100 chondroitin sulphate and keratan sulphate chains attached to different regions of the protein. Rotary shadowing has identified three globular regions in the proteoglycan (G1, G 2 and G3), G] at the N-terminal region contains the hyahironic acid binding region and G 3 displays some homology with a hepatic lectin. (B) Syndecan is a hydrophobic epithelial proteoglycan of composite structure, the core protein ( = 32 kDa) containing both heparan sulphate and chondroitin sulphate chains in the ectodomain. The glycanation pattern of syndecan varies according to the type of epithelium in which the protein is synthesised. (C) Decorin is a proteoglycan of the interstitial matrix that is firmly associated with collagen fibers. It contains only one glycosaminogiycan chain which is either chondroitin sulphate or dermatan sulphate, linked to an = 35 kDa core protein. (D) The secretory granule proteoglycan of haemopoietic cells contains a repeat sequence of serine and glycine in the core protein ( - 1 8 kDa) that allows high concentration of glycosaminoglycan chains to be assembled in a small peptide region. The proteoglycan contains either heparin or chondroitin sulphate. Details are provided in the text or in the following cited reviews: Refs. 5, 10, 14 and 24.

195 TABLE I

Disaccharide units of the glycosaminoglycans (A) Glycosaminoglycans are composed of disaccharide repeating units covalently linked to protein via the tetrasaccharide linkage sequence composed of xylose, two galactose residues and glucuronate. The glycosaminoglycans are attached to the amino acid serine in protein cores. (B) Chondroitin sulphate (CS) is made up of a disaccharide repeat of/31 ~ 4-1inked N-acetylgalactosamine (GalNAc)and /31 ~ 3linked glucuronic acid (GlcUA). The GalNAc residue can be sulphated at C-4 or C-6 to produce the type A or type C disaccharides. The polysaccharide chain usually contains both A- and C-type disaccharides although in haemopoietic ceils CS chains that contain only the A-type of disaccharide are quite common. A relatively high proportion of the E-type disaccharides (disulphated structure) are found in CS chains from mast cells and activated macrophages. Dermatan sulphate (DS) is also found in mast cells where it contains some disulphated, di-B-type disaccharides in addition to the more common B type, in which the glucuronate has been epimerised to iduronate (IdUA). DS chains always contain a variable quantity of CS-disaccharides. The disaccharide units in heparin and heparan sulphate consist of N-sulpho or N-acetylglucosamine (GlcNSO3 or GlcNAc) and GIcUA or IdUA. In heparin, trisulphated disaccharides containing GlcNSO3-6S and IdUA-2S are the most common structural unit. Heparan sulphate contains equal proportions of N-acetylated and N-sulphated disaccharides that are arranged in a predominantly segregated manner in the polymer chain. Hyaluronic acid is an unsulphated polysaccharide that is not covalently-linked to protein. The other sulphated glycosaminoglycans (GAGs) listed above are all covalently linked to serine residues in protein components to form the different classes of proteoglycan. (A) Linkage of glycosaminoglycans to protein serine-Xyl-Gal-Gal-GlcUA-(disaccharide repeat) (B) Disaccharide units (i) chondroitin sulphate

(ii) dermatan sulphate

Trivial name GalNAc-4S/31 --, 4 GIcUA type A GalNAc-6S/31 --* 4 GIcUA type C GalNAc-4S, 6S/31 ~ 4 GlcUA type E GalNAc-4S/31 ~ 4 IdUA type B GalNAc-4S,/31 --*4 IdUA-2S type di-B

(iii) heparin

GIcNSO3-6S al ~ 4 IdUA-2S

(iv) heparan sulphate

GIcNAc o.1-4GlcUA GIcNSO3 al-4 IdUA-2S

(v) hyaluronic acid

GIcNAc fll ~ 4 GlcUA

which b o t h a G l c N A c unit a n d the rare 3-0 s u l p h a t e g r o u p ( a t t a c h e d to G I c N S O 3 ) have b e e n i d e n t i f i e d [26]: GlcNAc-6S al ~ 4 GIcUA 131 ~ 4 GlcNSO3-3S, 6S al ---,4 IdUA-2S al ~ 4 GIcNSO3-6S It is likely that this p e n t a s a c c h a r i d e sequence is present in s o m e types of h e p a r a n s u l p h a t e [27,28]. However, the h e p a r a n sulphates are less s u l p h a t e d than h e p a r i n a n d

m u s t be r e g a r d e d as a s e p a r a t e family. In these glycos a m i n o g l y c a n s , there are a b o u t equal n u m b e r s of the N - a c e t y l a t e d a n d N - s u l p h a t e d disaccharides a r r a n g e d in a p r e d o m i n a n t l y segregated fashion [5,25]. The structure can be simply written as: (GlcNAc-GIcUA)n---(GIcNSO3-1dUA) m W h e r e n a n d m v a r y f r o m 2 - 9 a n d 2 - 8 , respectively. V a r i a t i o n s in the n u m b e r a n d d i s p o s i t i o n of ester s u l p h a t e g r o u p s in the N - s u l p h a t e d d o m a i n s ( T a b l e I) are largely r e s p o n s i b l e for the structural differences b e t w e e n h e p a r a n s u l p h a t e s [25]. In k e r a t a n s u l p h a t e the u r o n i c acid is r e p l a c e d b y galactose a n d the d i s a c c h a r i d e r e p e a t is G l c N A c / 3 1 4 G a l with s u l p h a t e g r o u p s at C-3 of galactose and, less c o m m o n l y , at C-6 of G a l N A c [29]. This p o l y s a c c h a r i d e has n o t b e e n i d e n t i f i e d in h a e m o p o i e t i c cells, b u t it seems to b e a c o m p o n e n t of the s t r o m a in r a b b i t b o n e m a r r o w [30]. T h e only g l y c o s a m i n o g l y c a n that does n o t c o n t a i n s u l p h a t e g r o u p s is h y a l u r o n i c acid which consists of a G l c N A c / 3 1 ---, 4 G l c U A d i s a c c h a r i d e repeat. T h e p o l y s a c c h a r i d e is synthesised at the p l a s m a m e m b r a n e b y a novel m e c h a n i s m which does n o t require direct assemb l y on a p r o t e i n core [31]. A l l the g l y c o s a m i n o g l y c a n s , especially h e p a r a n sulphate, show v a r i a t i o n in the degree a n d p a t t e r n of s u l p h a t i o n . T h o u g h we tend to refer to t h e m in singular fashion (i.e., c h o n d r o i t i n sulphate, h e p a r a n sulphate) they each c o n s t i t u t e g r o u p s of related structures, a n d the v a r i a t i o n s in c o m p o s i t i o n are often characteristic of the cell a n d tissue of origin. It w o u l d therefore b e m o r e a c c u r a t e to define g l y c o s a m i n o g l y c a n s as ' f a m i l i e s ' of c o m p l e x c a r b o h y d r a t e s which d i s p l a y p h y s i o l o g i c a l l y relevant p o l y m o r p h i s m s of m o l e c u l a r fine structure. Such v a r i a t i o n s are p a r t i c u l a r l y well c o r r e l a t e d with cell d i f f e r e n t i a t i o n in m a s t cells a n d m a c r o p h a g e s (see sections VI a n d IX).

H-D. Glycosaminoglycans of haemopoietic cells T h e g l y c o s a m i n o g l y c a n c o m p o n e n t s of h a e m o p o i e t i c cells have distinctive structural characteristics. F o r example, in h u m a n s the vast m a j o r i t y of cells in the p e r i p h e r a l b l o o d (e.g., l y m p h o c y t e s , g r a n u l o c y t e s , platelets, m o n o c y t e s , N K cells) c o n t a i n m a i n l y simple homopolymeric chondroitin-4-sulphates. Heteropolymers of 4- a n d 6 - s u l p h a t e d disaccharides, which are c o m m o n in o t h e r types o f m a m m a l i a n cell, are rare in b l o o d cells [32]. Basophils are e x c e p t i o n a l a m o n g s t the circulating cells b e c a u s e they synthesise c h o n d r o i t i n sulphates, e n r i c h e d in the t y p e E d i s a c c h a r i d e [33,34]. P e r i p h e r a l b l o o d cells in o t h e r m a m m a l i a n species generally c o n f o r m to the h u m a n p a t t e r n , synthesising

196 mainly homopolymeric chondroitin sulphates although heteropolymeric chains have been detected in murine B lymphocytes (see section X-B). It is also notable that a mouse pluripotential haemopoietic cell line (FDCP-mix) synthesises only chondroitin-4-sulphates [35]. In contrast with the uniform sulphation of the polysaccharides in peripheral blood cells, complex and highly acidic glycosaminoglycans are synthesised by bone marrow-derived cells that eventually reside in the tissues. Chondroitin sulphate E is produced by mucosal mast cells and macrophages, whereas connective tissue mast cells are the primary source of mammalian heparin (see Refs. 36 and 37, and section IX-B.2). These sulphate-rich glycosaminoglycans are likely to play important roles in the immune-related functions of mast cells and macrophages. II-E. Protein - glycosaminoglycan linkage

The disaccharide repeat sequences of glycosaminoglycans are not directly linked to protein. A tetrasaccharide sequence is present at the reducing end of the polysaccharide chains which terminates in a xylose residue. The xylose forms an O-glycosidic link to

pr-otei ,rl ER

serine in the core protein. The structure of the linkage region is: Ser-Xyl-Gal-Gal-GIcUA The substitued serine residues in the core protein are adjacent to glycine and the Ser-Gly dipeptide seems to be a basic requirement for recognition by xylosyl transferase enzymes. For several proteins which contain chondroitin sulphate or dermatan sulphate, including the invariant chain of class II antigens (see section IV-B), the glycosaminoglycan acceptor sequence has the general structure of: 'A'-Ser-GIy-'X'-GIyWhere ' A ' at the amino terminus is an acidic amino acid and ' X ' is unspecified. Synthetic peptides which contain this sequence are efficient xylosyltransferase substrates [38]. Ser-Gly acceptors for glycosaminoglycans also occur in other peptide sequences, although at least one acidic amino acid is often in close proximity. An interesting variation is found in collagen type IX in which chondroitin sulphate is linked to a serine residue with glycine at the N-terminal side, i.e., -Gly-Ser-AlaAsp [39].

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Fig. 3. Regulated and constitutive secretion of proteoglycans in haemopoietic cells. In the regulated secretory pathway (1) newly synthesised proteoglycans are stored in secretory granules and released only in response to specific stimuli. The proteoglycans are suggested to form an organised network inside the granule. In the constitutive pathway (2) proteoglycans are continuously secreted (2a), although the pathway is often associated with an alternative process in which proteoglycans are degraded to free glycosaminoglycanchains (2b). The chains are not secreted and eventually they are completely degraded in lysosomes. The diagram suggests the glycosaminoglycans may accumulate in a pre-lysosomal compartment where they could be bound to enzymes and basic proteins.

197 Potential Ser-Gly acceptor sequences have been identified for basement membrane and plasma membrane (syndecan) heparan sulphate proteoglycans from cDNA cloning [16,18]. The sequences are different from those in the majority of chondroitin sulphate proteoglycans because acidic amino acids are present at both the amino and carboxy terminals of the Ser-Gly dipeptide (e.g., Asp-Ser-Gly-Glu-Tyr) [18]. However, although this pattern may be a requirement for heparan sulphate synthesis, a recent paper on the amino acid sequence of a fibroblastoid proteoglycan named 'versican' indicates that the sequence is not specific for heparan sulphate [39a]. Versican only contains chondroitin sulphate chains, yet there are multiple copies of the syndecan-type sequence motif in the core protein. Therefore other characteristics of the protein structure must have an important bearing on the glycosaminoglycan composition of the proteoglycan. Furthermore, the cell type in which a protein is expressed can influence the glycanation pattern. This point is exemplified by proteoglycans in haemopoietic cells in which a common core protein can be subsitituted with heparin in connective tissue mast cells, chondroitin sulphate E in mucosal mast cells and basophils, or with chondroitin-4-sulphate in N K cells (section IX). III. Exocytosis and degradation of proteoglycans Control of the secretion of proteoglycans is central to their function in the haemopoietic system. Proteoglycan secretion and degradation have been studied in several cell types and two pathways of metabolic processing may be discerned. These can be defined in terms of whether secretion is regulated or constitutive (Fig. 3). In the regulated pathway, proteoglycans are stored in secretory granules and released in response to a specific stimulus. Secretion is regulated in mast cells (see, e.g., Ref. 40), N K cells, basophils, eosinophils and platelets. Constitutive (i.e., continuous) secretion occurs in cultured lymphocytes [41], monocytes and macrophages [42], although there is a variable proportion of proteoglycans that are retained inside the cells. The core proteins of these proteoglycans are eventually degraded and in some instances free chondroitin sulphate chains are detected within an intracellular, non-secretory compartment. Eventually, the chains are degraded and only rarely are they secreted from the cells. Pulse-chase analyses have clearly identified secretory and degradative pools of proteoglycans in FDCP-mix stem cells, human T-lymphocytes, a human monocytoid cell line, M1, and in HL-60 cells [35,43,43a,44,44a]. In FDCP-mix cells and T cells there was a lag of about 4 h between proteoglycan synthesis and the onset of degradation to free glycosaminoglycan chain~ [35,43a]. Differences in sulphation patterns were noted between the secretory and intracellular proteoglycans of M1 cells indicating

that a mechanism exists for sorting proteoglycan subsets [44].

IV. Protein cores

IV-A. Proteoglycans in secretory granules Detailed studies have been carried out on the major proteinase-resistant proteoglycans of mast cells, basophils, platelets and N K cells. The proteoglycans are located in secretory granules and they are released in response to specific stimuli. The distinguishing feature of the core protein is a long, repeating-sequence of serine and glycine in which each serine is a potential site for glycosaminoglycan synthesis. This sequence therefore enables glycosaminoglycans to be densely packed in a specific peptide domain. An ordered polymeric structure of this type was first proposed for heparin proteoglycan isolated from pronase digests of rat skin, and assumed to be produced by connective tissue mast cells [45]. The peptide core was highly enriched in serine and glycine and based on molecular weight parameters, the authors calculated that the proteoglycan ( M r > 900000) contained about 12 heparin chains ( M r 60000100000) attached to 2 / 3 of the serine residues in a speculated Ser-Gly repeat sequence. It was believed that this type of core protein might be unique to heparin proteoglycans. However, quite by chance, the first proteoglycan core protein to be cloned and sequenced was a relatively small molecule ( M r 18 600) which contained a sequence of 49 amino acids composed of alternate serines and glycines [21]. The proteoglycan was not isolated from mast cells but from a rat yolk sac tumour. When a partial c D N A clone for this proteoglycan (pPG-1) was used for Northern blot analysis of m R N A from a variety of rodent mast cells, N K cells and leukaemic basophils, a 1-1.3 kb mRNA was identified [46-48], indicating that these haemopoietic cells synthesise a protein component that is similar or identical to that found in the yolk Sac tumour proteoglycan. Molecular cloning studies have strengthened this view. The pPG-1 c D N A has been used to isolate full-length clones from a cDNA library of a rat basophilic leukaemia cell line (RBL-1). The concensus nucleotide sequence of two RBL-1 cDNA clones indicated that the coding regions are identical to the mRNAs produced by the yolk sac tumour, although a difference was noted in the length of the 5'-untranslated regions [48,48a]. This might indicate variations in control of m R N A translation. Recent studies have demonstrated that mouse and human haemopoietic cells produce a highly homologous protein to that found in the rat proteoglycan. In the mouse protein, identified from c D N A clones from normal and malignant mast cells, a Ser-Gly repeat sequence of only 21 amino acids constitutes the glycosaminoglycan attachment sites [49,50]. Proteoglycans re-

198 cognised by protein-specific antibodies contained heparin and chondroitin sulphate chains providing direct evidence for the broad glycosaminoglycan acceptor activity of the core protein [49]. The human analogue of the rodent proteoglycan gene was isolated from a cDNA library prepared from the promyelocytic leukaemia cell line HL-60. Analysis of the human cDNA clones predicted a protein of M r 17 600 which contains a serineglycine repeat sequence. In the human gene the length of this repeat is only 18 amino acids and it is interrupted by a single phenylalanine residue [51]. Close similarities were noted in the N-terminal sequences of the human and rodent peptide cores indicating that this region may be very important for the function of the proteoglycan. Human eosinophils also synthesise a similar core protein [52]. The molecular size characteristics of the eosinophil proteoglycan suggest that all serine residues in the Ser-Gly repeat are substituted with polysaccharide chains. This is a more efficient usage of serine acceptors than in the rat heparin proteoglycans [45]. The proteoglycan has been named 'serglycin' because of the distinctive Ser-Gly sequence in the protein core [14]. Although we still have no direct information on the amino acid sequences of the actual proteoglycans from human eosinophils or HL-60 cells, indirect confirmation that the predicted sequences from molecular cloning are correct has come from immune studies using an antipeptide antibody that recognised a specific sequence in the deduced amino acid sequence of the HL-60 derived cDNA [52a] and from studies of the purified proteoglycan from human platelets [53]. The first 66 N-terminal amino acids of the platelet proteoglycan have been sequenced and they are identical to the N-terminal region predicted from the cDNA for the mature HL-60 proteoglycan following signal peptide cleavage. It was also interesting that in about 10% of the platelet proteoglycan core proteins, the N-terminal domain of the mature protein had been cleaved at a site corresponding to a processing site noted in the yolk sac tumour proteoglycan [21]. From these important results, we can anticipate that purified granule-associated proteoglycans in human haemopoietic cells will be similar or identical to the protein component in the HL-60 proteoglycan. It has also been shown that the nucleotide sequence of cDNA from a library of acute myelogenous leukemia cells is identical to the platelet and HL-60 sequences, with the one exception of an additional 260 base pairs in the 3' untranslated region [54]. Quite recently the gene that encodes the core protein of the HL-60 proteoglycan was isolated and characterised [52a]. The whole gene is approx. 15 kilobases and contains three exons. The first exon codes for the untranslated 5'-region and the signal peptide of the translated protein containing 27 amino acids. The second exon codes for a 49 amino acid portion of the protein, presumably representing the

N-terminal part after processing in the endoplasmic recticulum. The last exon codes for the glycosaminoglycan attachment region preceded by a 17 amino acid stretch and followed by the C-terminal part of the protein and the 3' untranslated region of the transcript. In future studies it will be interesting to determine whether the serglycin core protein is found in mature blood cells that are not traditionally considered to store secretory material in cytoplasmic granules (see section VI and X). It may be significant however that normal human T cells constitutively secrete a pronase-resistant proteoglycan [43] and that a similar type of molecule has been identified in the pluripotential FDCP-mix cell line [35]. Finally, we will emphasise again that in rodents and humans different types of glycosaminoglycans (CS-E, CS-A and heparin) can be synthesised onto the Ser-Gly domain of the serglycine core protein. This implies that the primary amino acid sequence is not the main determinant of glycosaminoglycan structure. Perhaps proteolysis of the propeptide influences the glycanation pattern. Core protein isoforms, with subtle differences in glycosaminoglycan acceptor activities, could be produced by differential splicing of primary RNA transcripts, although no evidence for splicing has been obtained to date. Other factors that will influence glycosaminoglycan synthesis include the expression, activation and compartmentalisation of polysaccharide-synthetic enzymes. A particularly important control point will be the competing activities of GalNAc and GlcNAc transferases that transfer the amino sugar component of chondroitin sulphate and heparin, respectively, to 'primed' protein cores (i.e., proteins containing the tetrasaccharide linkage sequence of the glycosaminoglycans (see Table I)).

IV-B. Invariant chain proteoglycan in lymphocytes and rnacrophages The human class II antigens and their counterparts in mice (Ia antigens) are heterodimers composed of two polymorphic glycoproteins (a- and B-chains) whose structures are encoded by genes of the major histocompatibility complex [55]. They are expressed on Blymphocytes, monocytes and macrophages and they play a central role in immunoregulation by mediating the presentation of processed antigens at the cell surface. A third non-polymorphic component called the invariant chain (Ii) is often found in association with class II and Ia antigens [56]. It has been suggested that one of the functions of the invariant chain is to augment the antigen presenting capabilities of the class II molecules. An intriguing property of the li component is that it can be substituted with a single chondroitin sulphate chain to form a proteoglycan (Ii-CS) [57]. Site-directed mutagenesis has identified a serine residue at position

199 201 in a sequence of Ser-Gly-Leu-Gly as the site of GAG attachment [58]. The proteoglycan form of Ii can be co-immunoprecipitated with antibodies specific for a- or /3-chains so it seems to be in reasonably tight association with the class II complex [59]. Several cell types, including cell lines and freshly isolated cells, contain a proportion of their Ii (5%) as a proteoglycan [60]. There is some cell to cell variation in the length-of the CS chain, though accurate molecular weight data have not been published [61]. The function of Ii-CS is unclear. Although there is a relatively large intracellular pool of free, non-glycanated Ii [62], most of the Ii-CS is bound to the class II antigens and the trimeric complex is present inside the cell and on the cell surface. The association of Ii and class II begins in the endoplasmic reticulum (ER) [63,64] and the proteins undergo co-ordinate N- and O-glycosylation in the rough ER and Golgi with the chondroitin sulphate chains probably being synthesised on Ii in the medial-trans Golgi cisternae. About 5 min after Ii has been modified by the addition of chondroitin sulphate; the 'Ii-CS/class II' complex dissociates [60]. The reason may be that the glycosaminoglycan-bearing Ii regulates the transport of class II molecules to the subcellular site where antigen processing occurs [59]. It is conceivable that once the transfer step is complete, the class II must separate from the Ii-CS in order to combine with foreign proteins (please see Fig. 4, page 207). However, it is not known whether all class II antigens that are expressed on cell surfaces are transiently bound to Ii-CS [65]. Intracellular dissociation of the 'Ii-CS/class II' complex is apparently incomplete, as the complex has been detected on cell surfaces [60]. An obligatory role for Ii in the transfer of class II to the plasma membrane seems to be excluded because Ii-negative transfectants containing cDNA clones for class II will express class II antigens at the cell surface [66,67]. These studies did not address the question of the rate of transfer of class II molecules, nor their ability to bind and present antigens. Transfection studies with the site-directed mutation in the GAG-attachment site [58] should help to elucidate some aspects of the function of Ii-CS in the immune system. The discovery of this unique form of proteoglycan has been a most important and challenging development, and it is likely to reveal novel aspects of the biology of sulphated polysaccharides.

V. Granulocytes The granulocytes are generally divided into three distinct categories, depending upon cytochemical criteria. Accordingly, the cells are named eosinophilic, basophilic and neutrophilic granulocytes based on the appearance of their granules after staining [68]. The neutrophils are the most abundant of the three, representing approx. 60% of the total number of white blood

cells and more than 90% of the granulocytes. The predominant function of neutrophils is to kill bacteria. The eosinophils represent 1-3% and the basophils only 0.10.7% of the total number of white blood cells. The eosinophils are thought to be important in the immunity to helminth infections. The basophils are related to the mast cells, and both cell types are involved in allergic reactions. The developmental relationship between the two cell types is not completely clear. Most of the studies on granulocyte PGs have been performed on neutrophils (or polymorphonuclear granulocytes (PMN) as they are also more commonly described) and leukaemic basophils. Biochemical, autoradiographic and histochemical methods have been employed in the study of P G / G A G in these cells. Early studies revealed the presence of hexosamine- or alcian blue-positive material isolated from leukocytes, which comigrated with chondroitin sulphate (CS) standards after chromatography or electrophoresis [69,70]. Histochemical and autoradiographic studies indicated the presence of sulphated mucopolysaccharides in granules of neutrophils, basophils and eosinophils isolated from rabbit bone marrow. The same granules also contained basically charged proteins [71]. The presence of GAG, possibly CS and hyaluronic acid (HA), in the granules of rabbit PMN was confirmed by Fedorko and Morse [72]. Further isolation and characterisation of GAG from human granulocytes revealed that the hexosamine was mainly galactosamine, and infrared spectrum analyses demonstrated that the major glycosaminoglycan was chondroitin-4-sulphate (CS-A) [73]. Later, the availability of enzymes specifically degrading chondroitin sulphate and dermatan sulphate [74-76] allowed the demonstration of almost exclusively CS-A in human granulocytes [77]. Prior to this, metabolic studies showed that glycosaminoglycans were synthesised in granulocytes and stored as chondroitin sulfate proteoglycan [78] in association with basic proteins [79]. It was later suggested that the granules were lysosomes, and that chondroitin sulphate may interact with lysosomal enzymes and regulate their activities [80,81]. Detailed studies on the intracellular location of neutrophilic glycosaminoglycans have shown that they are present mainly in primary granules (lysosomes) with smaller quantities in secondary lysosomal granules which also contain lactoferrin and lysosyme [82-84]. The glycosaminoglycan content of secondary lysosomes may be underestimated because the tight complexation with basic proteins impairs their staining properties [85,86]. This possibility was suggested from studies on phagocytosis in which the glycosaminoglycan component of phagolysosomes was easily stained. Staining correlated with an increase in granule pH and the dissociation of the enzyme-glycosaminoglycan complexes [86]. Complexation and reversible inactivation of enzymes may be

200 one of the main functions of granular proteoglycans (see also section IX-D). PMN have also been demonstrated to release CS into the medium both during adhesion in vitro [87,88] and in conjunction with phagocytosis of bacteria [89]. Furthermore, a study comparing polymorphonuclear leukocytes isolated from peripheral blood and the peritoneal cavity showed that the former cells retained their GAGs, while they were secreted by the latter cell type [90]. Amongst the various types of granulocytes, glycosaminoglycans are most readily detected in basophils [91]. The highly sulphated polysaccharides (enriched in chondroitin sulphate E) are stored as proteoglycans in secretory granules [34]. The interactions and functions of basophil proteoglycans are broadly similar to the proteoglycans of mucosal mast cells, discussed in detail in section IX.

VI. Monocytes Monocytes and macrophages belong to the mononuclear phagocytic system which has several functions. The cells are specially adapted for phagocytosis and ingest particulate antigens. They also have an important role in the presentation of antigens to T and B cells. Finally, lytic activity is associated with the monocytoid lineage, the cells aquiring the ability to lyse tumor cells when activated by lymphocyte-derived mediators. Resting and stimulated monocytes and macrophages are highly secretory cells. Approx. 100 different secretory products have so far been identified [92]. Among these are CSPG, released from monocytes under in vitro conditions. The GAG chains are exclusively of the CS-A type [41,93]. However, when monocytes differentiate into macrophages in vitro (in the course of 5-7 days' incubation) the GAG chains become more highly sulphated, due to the appearance of approx. 20% of the E-type disaccharides with sulphate at C-4 and C-6 of GalNAc residues [93]. Such disulphated disaccharide units have also been demonstrated in cultures of mature human macrophages isolated from the peritoneal cavity [94]. The macrophages which develop from monocytes in vitro are cytotoxic to tumour cells. However, the culture substratum has a profound effect on monocyte differentiation. Monocytes cultured on collagen films, rather than directly onto plastic surfaces, do not become cytotoxic and appear more like the typical in vivo tissue macrophages [95]. Monocyte differentiation is inhibited on fibronectin substrata and the cells do not synthesise highly sulphated chondroitin sulphate [96]. It is conceivable that a plastic substratum induces the development of the in vitro counterpart to the (stimulated) foreign-body cells seen under certain pathological conditions in vivo. It has recently been demonstrated that monocytes stimulated after only 1 day in vitro with

a potent macrophage-activating agent (phorbol 12-myristate 13-acetate (PMA)) express oversulphated CSPG, with GAG structures identical to those expressed by in vitro macrophages. Furthermore, in macrophages differentiated from monocytes in vitro, the expression of disaccharides of type E was increased from 15 to 30% on a molar basis after phorbol ester treatment [37]. This is similar to the concentration of type E disaccharides in the chondroitin sulphates produced by mouse bone marrow-derived mast cells [36]. The increased expression of CS-E in the monocytoid lineage is therefore linked to the differentiation of monocytes into macrophages. In addition to this, it is interesting to note that only one of the typical monocyte/macrophage activators (PMA) induced the expression of CS-E in monocytes. Whether the phorbol ester mimics the effect of an unknown natural agent is at present not known. The fact that changes in the sulphation of the GAG chains of monocyte/macrophage proteoglycans are seen only under some defined experimental conditions might be helpful for identifying the biological correlate of this phenomenon. Significant amounts of CS-E have also been reported in guinea pig peritoneal macrophages stimulated with caseinate in vivo, whereas exposure of these cells to Escherichia coli in vitro did not lead to any changes in GAG structure [97]. The synthesis of CS-E by human macrophages does not seem to be dependent upon the core protein, because in monocytes [98] and also in mast cells [98a] the type E disaccharides are synthesised on fl-D-xylosides, which act as alternative primers for the synthesis of glycosaminoglycans. The biosynthesis of proteoglycans in monocytes has only been studied in in vitro systems, and under such conditions the CSPG is continuously released into the medium [41,42]. Recent studies may, however, indicate that the release of CSPG from a monocytic cell line may be adhesion dependent (Kolset, S.O., unpublished data). If this is a general phenomena one might perceive that circulating monocytes secrete CSPGs in order to modulate the extracellular (micro)environment when arriving in a specific tissue to become tissue macrophages, or when participating in inflammatory reactions. One study has suggested that peritoneal macrophages have been demonstrated to change the exposure of GAG on the cell surface during adhesion [99]. An early study indicated that macrophages were deficient in granule-associated glycosaminoglycans [72]. It was later shown by X-ray microanalysis that the monocytic cell line U937 does not contain proteoglycans in intracellular storage type granules [100]. Furthermore, the CSPGs released from cultured monocytes and macrophages bind fibronectin and collagen, the oversulphated species binding with the highest affinity [101]. Fibronectin is synthesised by macrophages [102] and, together with oversulphated CSPG, it may participate in the modula-

201 tion of the macrophage environment under normal and pathological conditions. The biological basis for the increased sulphation of the GAG chains related to differentiation and/or stimulation of monocytes remains to be established. Phenotypic changes in proteoglycan structure have also been investigated in conjunction with induced differentiation of the monocytic cell line U937 into macrophage-like cells. The latter cell type was demonstrated to express CSPG with GAG chains having a molecular mass of approx." 17 kDa, whereas the immature cells synthesised 30 kDa GAG chains [103]. The average molecular size of the GAG chains derived from the differentiated cells is closely similar to those found in CSPG synthesised by normal human monocytes [42]. Such defined structural changes may thus be used as a molecular parameter of differentiation along the monocytoid cell lineage. Macrophage proteoglycans may be involved in the development of arterial disease. Chondroitin sulphate proteoglycan secreted by a macrophage-like cell line (P 388Da) bound to low density lipoprotein (LDL) [104]. LDL-proteoglycan complexes are taken up by macrophages and this process may be related to the formation of foam cell macrophages (cells which have accumulated cholestrol esters) which are a feature of early atherosclerotic lesions. VII. Leukaemic cells in the myeloid series

Basophilic leukocytes isolated from two patients with chronic myelogenous leukaemia were demonstrated to contain exclusively chondroitin sulphate proteoglycan with a molecular mass of 750 kDa, whereas the GAG chains were in the order of 60 kDa molecular mass. Degranulation of the basophils led to the release of the intracellular CSPG [105]. The content of CS in the basophils was in the order of 5-12 gg per 106 cells, contrasted by 0.03 gg per 106 cells in the eosinophils [106]. In comparison, the content of heparin in rat peritoneal mast cells has been reported to be 26 gg/106 cells [107]. However, another study on basophils isolated from two patients with myelogenous leukaemia revealed the presence of chondroitin sulphate and significant amounts of heparin. The proteoglycan isolated had a molecular mass of about 140 kDa, with GAG chains of approx. 15 kDa [108] and was demonstrated to be pronase resistant. The presence of heparin in human basophils on the basis of susceptibilty of hexosamine-containing material to bacterial heparinase was also indicated in an earlier study [109]. Considering their GAG composition and the release of prostaglandins after activation (which is typical of mast cells and not basophils) these leukaemic basophils from two different patients may have differentiated into mast

cells, or the patients may contain both mast cells and basophils in the peripheral blood. The large differences, both in molecular weights and GAG structure of the proteoglycans from the two cited studies may be a phenotypic expression of heterogeneity within the leukaemic cell population. Furthermore, a rat basophil tumour proteoglycan was demonstrated to display a large polydispersity in molecular size and to carry predominantly CS and DS chains, although small quantitires of heparin were also detected [34]. It was later shown that the RBL-1 tumour actually synthesises a composite, proteinase-resistant proteoglycan in which short chondroitin sulphate and heparin chains (molecular mass 12 kDa) were attached to the same core protein [110]. The chondroitin sulphate chains comprised 73% of the total glycosaminoglycans and they were enriched in the di-B-type disaccharide (i.e., IdUA-2S fll--* 4 GalNAc-4S). The proteoglycans were deduced to be inside secretory granules because they were secreted by ionopohore-stimulated cells together with histamine and B-hexosaminidase. The common features of proteoglycans in mast cells (section IX) and basophils (high sulphation, proteinase-resistance, located in secretory vesicles) suggests close functional relationships between the macromolecules with sub-type specificities being generated by the different types of glycosaminoglycan and variations in their sulphation patterns. The expression of proteoglycan has been studied in the leukaemic cell line HL-60 in conjunction with the induced differentiation of these cells in vitro with agents such as retinoic acid, dimethyl sulfoxide or phorbol esters. The two former agents differentiate the cells into granulocytic cells and the latter leads to the development of macrophage-like cells. HL-60 cells synthesise CS-A, irrespective of inducer treatment [111]. Differentiation of the cells correlated with a decrease in the synthesis of GAG [111,112]. CSPGs synthesised by HL60 cells have a predominantly intracellular location and are resistant to proteinase treatment [113]. Changes in the turnover and sorting of GAG have also been related to the maturation of PMN on the basis of changes in the granule-association of radiolabelled sulphate [83]. One study has suggested that chondroitin sulphate is associated with the surface of mature granulocytes, whereas immature and leukaemic cells [114] do not express cell surface glycosaminoglycans. Further work is needed to confirm that proteoglycans are present on the granulocyte surface. Glycosaminoglycans both in the leukocytes and the stromal fibroblasts may possibly be important in the regulation of haemopoiesis. Recent studies strongly suggest that proteoglycans of the stromal matrix are important for the binding of haemopoietic growth factors, possibly in order to increase their local concentration and render them accessible to haemopoietic progenitor cells [3,4].

202 VIII. Platelets

Platelets play an important role in the regulation of hemostasis. Following injuries to the vessel wall the platelets adhere to subendothelial collagen, form aggregates in order to cover the lesion and release the content of their a-granules. These granules contain several bioactive molecules including, amongst others, serotonin, fibrinogen, platelet factor IV, ADP, thrombospondin, platelet-derived growth factor and proteoglycans. The proteoglycan content of platelets is mainly located in the a-granule component. The platelets also play an important role in the coagulation process, and an intimate relationship between platelet aggregation and blood coagulation has been established. As we discussed in section IV platelets contain the secretory granule proteoglycan, serglycin with chondroitin-4-sulphate being the major glycosaminoglycan component [115,116]. In early studies, a proteoglycan form of CS-A was isolated from human platelets and shown to have a molecular mass of 59 kDa [117], whereas a different experimental approach for the isolation of platelet PG, using platelet factor IV affinity chromatography as one of the isolation steps, resulted in the purification of a CSPG with a molecular mass of 53 kDa [118]. However, another attempt to isolate PG from platelets, using denaturing solvent and proteinase inhibitors (similar to conditions for the isolation of proteoglycans from cartilage) yielded a CSPG with a molecular mass of approx. 136 kDa, as determined by sedimentation equilibrium centrifugation [119]. The latter study was also performed in the presence of proteinase inhibitors and on freshly isolated platelets, whereas the work of Barber et al. [117] was done without proteinase inhibitors, and the work of Huang et al. [118] was performed on out-dated platelet-rich plasma. All three studies confirm the presence of exclusively CS-4 in human platelets. The molecular size of the CS chains appears to be approx. 28 kDa [119]. The N-terminal region of the peptide core of the platelet CSPG has been sequenced [53] and a more detailed presentation of these data was given in section IV. When platelets are stimulated with thrombin they release the content of their a-granules, and a secreted complex of PG and platelet factor IV has been recovered after such a stimuli [117]. However, a-granules of platelets have a complex composition and it is likely that the CSPG interact with more than one component. It is interesting to note that the a-granule proteins are arranged in an ordered manner with G A G as an important constituent [120,117], suggesting that the platelet CSPG is important for the organisation of intragranular structure. An inhibitor of the complement factor Clq has been demonstrated to be a chondroitin sulphate proteoglycan [121]. This particular proteoglycan, isolated from serum,

is very similar to the proteoglycan isolated from platelets [119]. Therefore, the Clq inhibitor may be derived from platelets. The biological implications, however, of the interactions between the platelet CSPG and platelet factor IV and the Clq inhibitor still remain to be outlined. The synthesis of platelet CSPG has been studied in vivo in guinea pigs and has clearly established that this proteoglycan is derived from the megakaryocytes. Furthermore, more mature megakaryocytes were demonstrated to synthesise a larger CSPG with larger G A G chains than the more immature cells [122]. In contradiction to human platelets the guinea pig platelets contain exclusively chondroitin-6-sulphated chains with molecular weight in the range of 40000-80000 [122]. It has been demonstrated that the synthesis of platelet factor IV and chondroitin sulfate by immature megakaryocytes was specifically increased by a megakaryocytestimulating factor isolated from thrombocytopaenic plasma [123,124]. These results might suggest that granule content and maturation in megakaryocytes is regulated by this specific factor. Additional information on proteoglycans (and sulphated glycoproteins) in platelets can be found in a review by Schick [125]. IX. Mast cells

Proteoglycans in mast cells have been more intensively studied than in any other type of haemopoietic cell. Historically, there are two main reasons for this: (i) mast cells produce heparin, an effective anticoagulant that has been in clinical use for over 40 years and (ii) granule metachromasia, a striking and characteristic property of mast cell cytoplasm, is due to the presence of highly sulphated proteoglycans. In recent years, progress in our understanding of mast cell proteoglycans has accelerated with improvements in methods for the isolation and culture of mast cells and with the application of recombinant DNA technology to the analysis of proteoglycan core proteins. These developments have led to a better understanding of both mast cell differentiation and of the relationship between proteoglycan synthesis and mast cell subtypes.

1X-A. Mast cell heterogeneity Mast cells are present in all organs and tissues of the body but in normal circumstances their numbers are relatively small. Most investigations have been carried out on mast cells from a restricted range of tissues in which they can be identified relatively easily. From these studies, two basic classes of mast cell are recognised in rodents, these being the mucosal mast cells (M-MC) and connective tissue mast cells (CT-MC) [126]. By definition both sub-types will bear IgE-Fc receptors

203 TABLE II Differences between mucosal and connective tissue mast cells

Mucosal

Connective tissue

Low histamine content

High histamine content

Short life-span (7"1/2 approx. 40 days)

Long life-span ( T1/2 > 6 months)

Thymus dependent proliferation in immune response

No proliferative immune response

Chondroitin-4-sulphate Chondroitin-4,6-disulphate

(CS-E) Chondroitin sulphate type B

Heparin

Proteinase II (rat)

Proteinase I (rat), caroboxypeptidase Many granules, uniform, large size

Few small granules of variable size

See review by Enerb~ick [126] for further details.

and secrete histamine following receptor activation. As their names suggest, one of the distinguishing characteristics between these two sub-classes of mast cell is their anatomical location, M-MC residing in the lamina propria of the intestinal epithelium and the CT-MC being present in skin and the serosal lining of the peritoneal cavity. There are also morphological and biochemical criteria by which these cells can be distinguished (Table II) and these include differences in number and electron density of the secretory granules, histamine content, proteinase composition and, most significantly for our present discussion, in the concentration and structure of the granule proteoglycans. It was once believed that all mast cells contained heparin, but the differences in binding of metachromatic and fluorescent dyes between mast cells from separate anatomical sites, gave the first suggestion that proteoglycans were variable constituents of the mast cell [127]. These findings have been confirmed by detailed biochemical analysis.

IX-B. Glycosaminoglycan components of mast cell proteoglycans IX-B. 1. Mucosal mast cells (M-MC)

Actively proliferating cultures of rodent mast cells can be produced by maintaining cell suspensions from haemopoietic tissues in the presence of multi-lineage growth factor, interleukin-3 (IL-3), or by using T-cell conditioned medium as a source of IL-3. The mast cells produced in such cultures resemble mucosal mast cells in their histochemical and biochemical properties (Table II). These cultures of mucosal-like mast cells synthesise chondroitin sulphate E chains containing 60-70% of 4-sulphated disaccharides (CS-A) and 30-40% of the E-type disaccharides (GalNAc-4S, 6S, /31 ~ 4 GlcUA;

Table I). The chondroitin sulphate chains are relatively small (M r 25 000) and approx, eight chains are present in the CSE-PG of M r 200000-250000. Intracellular degradation to produce detectable levels of free GAG chains does not occur. The proteoglycan is stored in secretory granules and released in response to activation of the IgE-Fc receptor [128]. It has not been possible to isolate sufficient quantities of M-MC from normal tissues to compare their proteoglycans with counterparts produced by cultured cells. In helminth-infected rats however, there is a large T-cell dependent proliferation of M-MC in the intestinal epithelium. The proteoglycans produced by these cells have been studied following metabolic labelling in vivo with [35S]sulphate [129,130]. The isolated 35S-macromolecules contain highly sulphated chondroitin sulphate chains [129,130]. The first studies on their sulphation patterns indicated that disulphated disacchatides obtained after chondroitin sulphate lyase digestions consisted mainly of the type E structures [129]. A novel methodological approach did, however, lead to the identification of both type E and type B disaccharide units in a ratio of approx. 1:1 [131]. Disulphated disaccharides accounted for approx. 40% of the total amount of disaccharides, the other 60% being the type A (i.e., 4-sulphated) units [131]. However, using different analytical methods, the work of Stevens and coworkers [130] suggested that the major disulphated disaccharide is the di-B component (GalNAc-4S 131 ---,4 IdUA-2S) with only minor quantities of the E-type disaccharide (7%) being present. Some of the structural differences could be due to different strains of rats used by the two groups and perhaps also to unrecognised variations in the characteristics of the helminth infections. Consideration should also be given to the possibility that atypical chondroitin sulphate isomers might be synthesised by M-MC from infected animals because standard degradation procedures with condroitin sulphate lyases gave relatively low yields of disacchatides [131]. IX-B.2. Connective tissue mast cells (CT-MC)

The only proteoglycan synthesised by isolated rodent peritoneal CT-MC is the heparin proteoglycan [132]. Ten to twelve large heparin chains (M r 70000-80000) are present in the proteoglycan of M r 750 000-900000. As indicated earlier, the CT-MC contain a high concentration of heparin, (15-50 /~g of proteoglycan per 10 6 cells) stored in electron dense secretory granules which are a dominant cytological characteristic of the cytoplasm [133]. The PG content is much higher than in the M-MC which contain 1.5-2.3 txg of proteoglycan per 106 cells [36]. Heparin is the most polyanionic macromolecule in mammalian tissues containing one carboxyl group and an average of 2.4 sulphate groups for each disaccharide. Under normal circumstances it is

204 a unique CT-MC product and this presumably indicates that there are discrete and specialised functions for the CT-MC in which heparin plays an integral role. Heparin proteoglycan synthesised by cultured rat peritoneal mast cells is polydisperse, and this is due to the presence of different numbers of heparin chains in the macromolecules rather than to variations in heparin chain length [134]. Heparin oligosaccharides are not detected in the cell cultures. This is in striking contrast to anticoagulant heparin prepared from bovine lung or pig mucosa which mainly consists of heparin fragments in the Mr range 8000-25000 [45]. Some degradation could occur as a result of post-mortem change in the tissues or perhaps during individual extraction procedures. However, it seems that partial depolymerisation of heparin is a normal physiological process caused by a widely-distributed endoglycosidase which attacks heparin at selected glucuronic acid residues [135,136]. Heparin degradation by endoglycosidase in rat mastocytoma cell cultures is elicited by contact with macrophages [136a]. The macrophages might be the source of the enzyme or they could activate latent enzyme in the mastocytoma cells. On the basis of these and other results a two-stage degradation scheme has been proposed for heparin proteoglycan in mastocytoma tissue in which a specific proteinase releases heparin chains from the core protein and the free chains are then degraded to oligosaccharides by the endoglycosidase [136a]. It remains to be established that this model is applicable to normal mast cells. Heparin metabolism has been investigated in rats following intraperitoneal administration of [35S]sulphate [137]. A wide range of [35S]heparin depolymerisation products were isolated, heparins from the lung and peritoneal cavity were extensively degraded, while heparin from the skin was largely in the form of a proteoglycan. The findings support the view that the formation of heparin oligosaccharides is a normal process and tissue-to-tissue variations in molecular size could be due to differences in heparin structure (i.e., concentration of endoglycosidase cleavage sites) and/or the availability of macrophages. Although heparin is the only proteoglycan synthesised by rat CT-MC in culture small quantities of chondroitin sulphate E have been detected in the secretory granules [138]. Since mature M-MC synthesise CS-E and can be induced to differentiate to CT-MC in vitro and in vivo (see section IX-C) the granule chondroitin sulphate E could reflect an earlier M-MClike stage of development. However, an alternative possibility must be considered. A latent capacity for the synthesis of chondroitin sulphate E in peritoneal mast cells was revealed by addition of fl-D-xylosides [139,140]. The mast cells formed E-type chains, but no heparin, on the fl-D-xyloside primer. There may be special circumstances in vivo, e.g., bacterial infection or tissue

damage, in which CT-MC are induced to synthesise chondroitin sulphate E as well as heparin. IX-B.3. Human mast cells

The existence of mucosal and connective tissue phenotypes in human mast cells is suggested by studies on proteinase distribution [141] and histochemical criteria [142]. There have been relatively few investigations of the proteoglycans in human mast cells. Mucosal mast cells in human intestinal biopsies synthesise chondroitin sulphate E, with 10% of the type E disaccharide [143], but the histochemical characteristics of mast cells from the nasal mucosa suggested that the major glycosaminoglycan may be heparan sulphate [144]. In the same study heparin was identifed in human skin mast cells and 35S-labelled heparin has been separated in skin biopsies from a patient with urticaria pigmentosa [145]. Highly purified preparations of human lung mast cells synthesise heparin and chondroitin sulphate E in a ratio of 2 : 1, the E-type disaccharides comprising 10-25% of the chondroitin sulphate chain [146,147]. The two glycosaminoglycans were attached to separate, proteinase-resistant core proteins. The chain length of the heparin was only 20K, significantly shorter than the heparin chains produced by rodent CT-MC [148]. It is not known whether two sub-groups of mast cells are present in the lung preparations which synthesise different proteoglycans. Various lines of investigation indicate that human lung mast cells cannot be readily assigned to the mucosal or connective tissue categories and they could represent a discrete population. The relationship between the production of different types of proteoglycan and other biochemical and morphological parameters may eventually help to define the spectrum of mast cell phenotypes in human tissues. Heparin with anticoagulant activity has been isolated from the spleen of a patient with malignant mastocytosis [149], but in this study chondroitin sulphate was enzymatically removed during the isolation procedure. IX-C. Proteoglycans and mast cell development

New ideas have emerged in recent years on the developmental relationships in rodent mast cells. If cloned, IL-3 dependent, bone marrow-derived mast cells (BMMC), with some characteristics of M-MC are transferred to a feeder layer of 3T3-fibroblasts, the cells will adhere to the fibroblast monolayer and a proportion of the adherent cells will undergo a phenotypic conversion to connective tissue-like mast cells [150]. The adherent cells increase their histamine content, produce larger numbers of secretory granules which counterstain red with Safranin, and they begin to synthesise heparin proteoglycan. Mast cell differentation requires direct contact with the fibroblasts and mast cells remain via-

205 ble, though non-proliferative, in the absence of IL-3. Cells with intermediate staining characteristics between BMMC and CT-MC were also observed. The ability of cultured M-MC to differentiate into CT-MC has also been demonstrated in genetically defective mice which do not contain mast cells [151]. Injection of BMMC into these mice leads to the appearance of characteristic CT-MC in the peritoneal cavity. These studies indicate that there is likely to be a common progenitor for mast cells and that the microenvironment plays an important role in determining the cellular phenotype. The results also suggest that the BMMC could be an intermediate in the normal pathway of differentiation to both M-MC and CT-MC, but the possibility remains to be firmly established. A significant advance in mast cell culture was reported recently [152] when cultures of connective tissue-like mast ceils were established by co-culturing splenocyte-derived progenitor cells with fibroblasts that produce a murine sarcoma virus containing the K-ras oncogene. The resulting immortalised mast cells were infected with the virus and grew without addition of IL-3. Sub-clones were isolated that synthesised mainly heparin proteoglycan. K-ras transfection also induced the formation of mast cell cultures with intermediate phenotypes, displaying both CT-MC and M-MC characteristics. The results give further support to the view that a spectrum of mast cell subsets may be present in normal tissues even though the in vivo picture is dominated by the two major populations, the M-MC and the CT-MC. This result is consistent with the data acquired from seeding M-MC onto inductive fibroblast monolayers. Intermediate forms could be difficult to recognise in tissues because of their low abundance. IX-D. Functions of mast cell proteoglycans

Many suggestions have been put forward for the biological functions of the proteoglycans in mast cells, some of the propositions are backed up by experimental evidence, others are mainly speculative and largely based upon the high sulphation of the glycosaminoglycan chains. A fundamental issue is whether the proteoglycans have defined roles both within the secretory granules and following release into the extracellular space. The prevailing evidence indicates that they are important for the efficient concentration of secretory material in granular structures (analogous to the function of proteoglycan in platelet a-granules) and for mediating the physiological responses of mast cell activation. The cytoplasmic vesicles of CT-MC contain the basic proteinases chymase and carboxypeptidase. Heparin proteoglycan has been identified in complexes with these enzymes in surface invaginations of the discharged

cell [153], and this probably reflects their aggregated state in secretory vesicles. Proteoglycan complexation with proteinases will restrict enzyme diffusion (possibly confining their activities to the immediate vicinity of the mast cells) and perhaps facilitate the rapid co-ordinated breakdown of protein substrates. [3H]DFP labelled serine proteinases from cultured M-MC have also been detected in macromolecular complexes with proteoglycans, but only a small quantity of the cellular proteoglycan was found in the aggregates. An unexpected finding was that the complexed proteoglycan contained both chondroitin sulphate E (characteristic of M-MC) and heparin, the two types of chains being present predominantly on separate core proteins [154]. Although heparin comprised only 3% of the M-MC proteoglycan, 60% of the proteoglycan in the complex was heparin. This preferential association strongly suggests a physiologically relevant affiliation. In addition to proteinases, the other major basic substance in mast cell granules is histamine, the physiological mediator of inflammatory reactions. The highest concentrations of histamine and proteoglycan (heparin) are found in the CT-MC (Table II). The polyanionic properties of heparin will clearly help to maintain electrical neutrality in histamine-rich secretory vesicles. It has also been suggested that in the secreting cell, the hydrophilic proteoglycans could accelerate osmotic swelling prior to granule fusion with the plasma membrane [153]. The ordered polymeric structure of mast cell proteoglycans should facilitate the formation of the organised granule matrix suggested by the high electron density and crystalline arrays observed in secretory vesicles of CT-MC. The influence of heparin in the regulation of blood coagulation remains a puzzle. It seems likely that the presence of unique antithrombin-binding sites in heparin should indicate functions in the control of blood coagulation. Heparin proteoglycan has been demonstrated to have an assymmetric distribution of GAG chains with high affinity for antithrombin [156,156a], which suggest that there is a division of function between the high affinity chains possibly destined to participate in the regulation of coagulation, and the low affinity chains synthesised for other purposes. Heparin is not found in the bloodstream in normal circumstances, but a transient presence in inflammatory conditions cannot be excluded. One plausible suggestion is that heparin inhibits fibrin formation at extravascular sites both by accelerating antithrombin binding with thrombin and by inhibiting the formation of the prothrombinase complex on the surface of activated macrophages [157]. It was recently shown that clams contain heparin with high affinity antithrombin binding sequences [158]. Clams do not have a blood circulation system and attempts to isolate antithrombin-like proteinase inhibitors from clams were unsuccessful.

206 The partial degradation of heparin chains that occurs in many tissues is an intriguing metabolic characteristic (section IX-B.2) of uncertain physiological relevance. It is not known whether the heparin-oligosaccharides are distributed to the regulated secretory pathway in CT-MC (Fig. 3) or whether they represent intermediates in a normal degradation process. The tissue endoglucuronidase that degrades heparin does not attack the glucuronic acid in the antithrombin-III binding sequence [136], suggesting that the preservation of this sequence is an important property for the derived oligosaccharides. It is possible that released heparin oligosaccharides are important for inhibiting fibrin formation in tissues, whereas the heparin proteoglycan has the major functions of granule matrix organisation and the regulation of proteinase activity at cell surface.

X. Lymphocytes There are two basic types of lymphocyte, the Tlymphocytes which mature in the thymus gland and B-lymphocytes which develop in the bone marrow. Although T and B cells are derived from a common progenitor cell (Fig. 1), they have separate but interdependent functions in the immune system [158a]. B cells are the antibody producing cells and are responsible for humoral immunity. The proliferation of B cells requires interaction with a subset of T cells, the so-called T-helper cells, whereas a different T-cell subset, the T-suppressor cytotoxic cells, regulate the magnitude of the immune response and eliminate cells infected with viruses when virus-specific antigens are expressed on the cell surface. Plasma membrane antigens CD-4 and CD-8 are expressed on T-helpers and T-suppressors, respectively, and may be used for cell sorting or phenotypic analysis of T-cell clones [158b].

X-A. T-lymphocytes There have been few studies on lymphocyte proteoglycans. Early reports indicated the presence of chondroitin sulphate in inclusion bodies of guinea pig lymphocytes [159]. Mitogen-stimulated human T cells produce chondroitin-4-sulphate proteoglycan [43] which is rapidly secreted into the culture medium, rather than being stored inside the cells (see Ref. 43a, and Fig. 3). They therefore fall into the category of constitutive secretory cells, and in common with other haemopoietic cells of this type not all the proteoglycan is secreted and a variable proportion is converted to free chondroitin chains. These chains are retained inside the cell and are eventually degraded in lysosomes. When separate clones of T-helper and T-suppressor cells were examined, unexpected but reproducible differences were observed in proteoglycan metabolism even though the structure of the macromolecules was the

same in both groups. The CD-4-positive T-helper cells were more active in the secretion of proteoglycan releasing about 5-fold more material into the culture medium than T-suppressors over a 48 h incubation period. There were also differences in the amounts of chondroitin sulphate present in the cells as proteoglycan or free glycosaminoglycan. The T-helpers contained mainly proteoglycan (ratio of chondroitin sulphate in proteoglycan/free glycosaminoglycan was 3:1), whereas the corresponding ratio in suppressor/cytotoxic cells was about 1:1. These differences were observed in T-cell subsets from six different individuals suggesting that proteoglycan metabolism is directly linked to the specific immune functions of T-cell subsets (Steward, W., Christmas, S., Lyon, M. and Gatlagher, J.T., unpublished data). It is possible that proteoglycans act as vehicles for the intracellular transport and release of regulatory molecules acting on B cells. It has also been suggested that T-cell proteoglycans are directly mitogenic for B cells [160], but since B-cell growth factors are active in the picomolar range the purity of the proteoglycan needs to be thoroughly tested. Chondroitin sulphate and small quantities of heparan sulphate are produced by mouse thymic lymphocytes [32]. Proteoglycan sythesis is significantly increased in such cells by the lymphokines IL-1 and IL-2, and the effect is not dependent upon cell proliferation [161]. The secretion of chondroitin sulphate proteoglycan was considerably enhanced in a metastatic variant of a mouse T-lymphoma cell lines - Eb [162]. The authors suggest that the proteoglycan could have a direct influence on the malignant properties of the cells by modifying cell adhesion or by promoting the activities of autocrine growth factors. A minor population of lymphoid cells in the peripheral blood are the so-called natural killer or NK cells which have some T-cell-like characteristics [163]. These cells may play an important role in immune surveillance against cancer [164]. The proteoglycans (containing chondroitin-4-sulphate chains; Ref. 43) are located in secretory granules along with cytotoxic effectors such as perforins, serine proteinases and cytolysins [165-167a]. These molecules are believed to be reversibly inactivated by binding to proteoglycans in the low pH of the secretory granule [168]. When the cell is activated, the granule contents are released onto the surface of an appropriate target cell and the complexes dissociate in the ambient pH environment leaving the toxic mediators to attack the target cell membrane. The dissociated proteoglycan could protect the NK cell surface from attack by the cytotoxic agents.

X-B. B-lymphocytes The differences in sulphation patterns in chondroitin sulphate from mouse T- and B-lymphocytes was dis-

207 cussed in section III and the structure of the Ii proteoglycan of B cells was described in section IV-B. In additon to the Ii protein another family of lymphocyte membrane proteoglycans that appear as glycanated variants are the Hermes cell surface antigens [169]. The glycosaminoglycan component is chondroitin sulphate. These antigens are called homing receptors because they mediate the binding of B- (and T-) lymphocytes to high endothelial venules (HEV) and therefore play an important role in lymphocyte migration from blood to lymphatic tissue. Chondroitin sulphate may interact with accessory sites on HEV or stabilise an appropriate conformation of the Hermes antigen binding site. Recently it has also been shown that syndecan (see Fig. 2) is expressed on the surface of B-lymphocytes during defined maturation stages when the cells interact with extracellular matrix. Circulating B cells do not express syndecan, which suggests that the proteoglycan may play a role in the adhesion of the cells at specific stages of the B cell development [170].

XI. Summary Proteoglycans are produced by all types of haemopoietic cells including mature cells and the undifferentiated stem cells. The proteinase-resistant secretory granule proteoglycan (serglycin; Ref. 14), is the most prevalent and best characterised of these proteoglycans. Although its complete pattern of distribution in the haemopoietic system is unknown, serglycin has been identified in the mast cells, basophils and NK cells, in which secretion is regulated, and in HL-60 cells and a monocytoid cell line (Kolset, S.O., unpublished data) in which secretion is constitutive. Proteinase-resistant proteoglycans have been detected in human T-lymphocytes and murine stem cells (FDCP-mix) and the core proteins may be closely related to serglycin. A variety of glycosaminoglycan chains are assembled on the serglycin protein and it is likely that this class of proteoglycan can carry out a wide variety of functions in haemopoietic cells including the regulation of immune responses, inflammatory reactions and blood coagulation. There is strong evidence that in mast cells, NK cells and platelets, the proteoglycans are complexed to basic proteins (including enzymes and cytolytic agents) and amines in secretory granules and such complexes may dissociate following secretion from the cell. The stability of the complexes may be regulated by the ambient pH which may be acidic in the granules and neutral or above in the external medium. However, proteinase-proteoglycan complexes in mast cell granules seem to remain stable after secretion and it has been proposed that the proteoglycan regulates activity of proteinases released into the pericellular domain. The functions of proteoglycans which are constitutively secreted from cells are less clear. If cells have no

requirement for storage of basic proteins why do they utilise the same design of proteoglycan as cells which accumulate secretory material prior to regulated release? We should stress that the so-called constitutive secretory pathway has been identified in haemopoietic cells in culture, which are usually maintained and grown in the presence of mitogenic factors (e.g., IL-2, IL-3). The cells are therefore activated and it has not been established that continuous proteoglycan secretion occurs in quiescent cells circulating in the peripheral blood. It is possible that lymphocytes, monocytes and macrophages, in which the constitutive secretion pathway operates in vitro, may store proteoglycan in vivo unless stimulated by mitogens or other activating agents. Alternatively, these cells may not store their proteoglycans, but have a low level of continuous synthesis and secretion in vivo, which may be shifted towards higher levels of metabolic activity when the cells are activated. The secreted proteoglycans could bind and 'condition' the extracellular matrix of tissues in which these cells are activated (e.g., lymph nodes, sites of inflammation, damaged blood vessels) or they could function as carriers for cytokines or other agents that regulate the functions of adjacent cells. The constitutive secretory pathway is often associated with the degradation of a proportion of proteoglycan to free glycosaminoglycan chains. This intracellular pool may represent the sulphated components identified in neutrophils in early histochemical studies (section V) and believed to be located in lysosomes. Since glycosaminoglycans are degraded in these organelles it is possible that they are held in a specialised lysosome population (pre-lysosomes, Fig. 3) in which glycosidase activities are suppressed. The polysaccharides are probably complexed with lysosomal enzymes and, in a similar fashion to proteoglycan-enzyme complexes in secretory granules, pH may be one of the

~-

Ii-CS

Class II Acidification and Dissociat i on

,

Proteolysi s

"

~

~ ~. Cell surface

ag

R+ ag ag

Association

Presentation

Fig. 4. Role of invafiant-ch~dn proteoglycan (Ii-CS) in the function of class II antigens: convergence of pathway of dissociation with antigen (Ag) processing and presentation. CS chains are synthesised onto Ii in the Golgi. The CS component may assist the transfer of 'class II/Ii-CS' to an acidic vesicle, possibly a recycling endosome carrying incoming antigen (Ag) complexed to antigen receptors (R). Acidification of the endosome activates proteolytic cleavage of Ag to antigenic peptides (ag) and dissociation of the class II/Ii-CS complex. Class II are then free to bind ag, and the new complexes are presented at the cell surface. R, receptor; Ag, antigen; ag, processed antigen.

208 factors that determines the stability of the interactions. The question of why immature cells, including multipotent stem cells (FDCP-mix), synthesise proteoglycans is unresolved. Stem cells store the majority of newlysynthesised proteoglycan, even in the continuous presence of the growth factor IL-3, and agents that induce secretory activity have not been found. The small, but important, minor proteoglycan species in lymphocytes (Hermes homing receptor antigen, Ii chain, syndecan) were identified by immunological methods, Hermes and Ii occurring as glycanated variants. These proteoglycans would have been difficult to identify as discrete species without the use of antibody reagents. They are especially interesting because they strongly suggest that the glycosaminoglycan chains will have a major influence on lymphocyte migration (Hermes), adhesion (syndecan) and antigen processing (Ii), activities which are central to normal lymphocyte function. Future studies should provide new insights on the molecular structure of the minor and major proteoglycan components in the haemopoietic system, and it likely that novel ideas will emerge on how proteoglycans influence the development and biological properties of this diverse and widely disseminated class of cells.

Acknowledgements We thank the Cancer Research Campaign (U.K.) and the Norwegian Cancer Research Foundation for financial support, and Dr. Richard Stevens for his very helpful comments on the manuscript.

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Proteoglycans in haemopoietic cells.

Proteoglycans are produced by all types of haemopoietic cells including mature cells and the undifferentiated stem cells. The proteinase-resistant sec...
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