Clin Exp Immunol 1996; 104:13

EDITORIAL REVIEW

Glycosaminoglycans contribute to multiple functions

of vascular endothelial cells

P. COCKWELL, D. H. ADAMS* & C. O. S. SAVAGE Renal Immunobiology Group, Centre for Clinical Research in
Immunology and Signalling (CCRIS), The Medical School, University of Birmingham, and *Liver Laboratory, Clinical Research Block,
Queen Elizabeth Hospital, Birmingham, UK

(Accepted for publication 9 January 1996)

Proteoglycans (PGs) are a group of complex macromolecules that
may have different GAG
are expressed in all tissues and play a pivotal role in cell function
and connective tissue formation [1]. They are found within the cell
plasma membrane, the basement membrane and the extracellular
matrix (ECM). In addition they are present in intracellular loca-
tions such as secretory granules. PGs consist of a core protein
(which contains a membrane spanning domain in cell surface-
associated molecules), to which one or more glycosaminoglycans
(GAGs) are covalently attached. There are four types of GAGs:
chondroitin sulphate, heparan sulphate (HS), dermatan sulphate
and keratan sulphate, and one usually predominates on any
particular PG family. Distinct protein cores and marked hetero-
geneity in the structure of the GAG side chains contribute to the
diverse biological role of PGs [2]. In addition to providing a
scaffold for ECM, PGs play a vital role in sequestering and
presenting a wide range of growth factors and cytokines to a
diverse range of responding cell types, including leucocytes and
the endothelium.
PGs, most of which are HSPGs, are expressed on both the
luminal and basal endothelial cell (EC) surface, where they are
uniquely situated to modulate the vascular microenvironment by
regulating leucocyteEC interactions, growth factor-receptor pre-
sentation, coagulation and vascular permeability. On page 60 of
suggesting that TNF- and IFN-
binding preferences or affinities.
The ability of PGs to bind and present cytokines and growth
factors was first suggested by Gordon et al. in 1987, who described
the need for PG presentation of IL-3 in bone marrow stroma [4].
Studies on basic fibroblast growth factor (bFGF) then demonstrated
a necessity for PG for efficient engagement and activation of the
high-affinity FGF receptor [5]. With the realization that many
inflammatory cytokines contain GAG binding domains, this para-
digm has been extended to include the presentation of chemokines at
the EC surface.
The studies of Tanaka et al. suggest that interactions with
GAGs have a pivotal role in modulating the presentation of
chemokines to circulating leucocytes [6]. Chemokines are a
family of small, heparin-binding cytokines that are produced by
multiple cell types on activation [7]. They are involved in the
recruitment of circulating leucocytes to tissue through chemo-
attraction and by facilitating integrin-mediated binding to endo-
thelium [8,9]. Chemokines are divided into three subfamilies based
on the number and position of their cysteine residues [10]. Two of
the families comprise molecules with four cysteine residues that
form two disulphide bonds: the C-X-C or  chemokines, where the
first two cysteine residues are separated by an amino acid, and the
this issue Rix and colleagues identify a further role for endothelial C-C or  chemokines, where they are adjacent. The third family,
GAGs in modulating the proinflammatory effects of interferon- known as C chemokines, have only two cysteine residues and one
gamma (IFN-
) [3]. They show that soluble GAGs, particularly disulphide bond. These structural distinctions determine functional
heparin, can inhibit the ability of IFN-
to stimulate ECs to express specificity; the C-X-C chemokines predominantly act on neutro-
MHC class II antigens and up-regulate intercellular adhesion phils, the C-C chemokines on monocytes, eosinophils and lym-
molecule-1 (ICAM-1) in vitro. They suggest that soluble GAGs phocytes, and the C chemokines on lymphocytes. There is
interfere with IFN-
binding to EC surfaces, indicating a require- evidence that chemokines from both the C-X-C and C-C families
ment for the presentation of IFN-
to its high-affinity receptor by bind to the EC surface, where they are ideally placed to activate
EC surface-associated GAGs. The functional significance of these leucocytes. For example, Rot demonstrated that the C-X-C che-
observations was confirmed by demonstrating that heparin inhibits mokine IL-8 binds to the luminal surface of post-capillary venules
the binding of activated T cells to EC in vitro. Interestingly, and small vein endothelium [11], the site of leucocyte transmigra-
heparin was unable to abrogate the effects of tumour necrosis tion in most inflammatory states; whether this binding was speci-
factor-alpha (TNF-), which also has a GAG binding potential, fically linked to GAGs was not studied, although the kinetics and
Correspondence: Dr Caroline O. S. Savage, Renal Immunobiology affinities are compatible with retention in the glycocalyx. In
Group, Centre for Clinical Research in Immunology and Signalling addition, in vitro studies have shown that the C-C chemokine
(CCRIS), The Medical School, University of Birmingham, Edgbaston, macrophage inflammatory protein-1 beta (MIP-1 ) immobilized
Birmingham B15 2TT, UK. on proteoglycan is equally effective as soluble chemokine in
# 1996 Blackwell Science 1
2 P. Cockwell, D. H. Adams & C. O. S. Savage
activating T cell adhesion to the integrin ligand vascular cell
adhesion molecule-1 (VCAM-1). Several PGs were used in these
studies, including HSPG, heparin and the PG form of CD44 [12].
GAGchemokine interactions are likely to be complex, with
the potential for providing both site- and chemokine-specific
presentation at the vessel wall. This is because GAGs display
differential specificity for structural motifs, implying the potential
for differential binding between locally produced chemokines and
GAGs which is dependent on the specific inflammatory trigger and
binding sites between cytokines and growth factors may modulate
the local inflammatory response. Other growth factors may also
have a requirement for cell surface-associated heparin-like
molecules; vascular endothelial growth factor (VEGF), for
example, binds heparin through an epitope that appears distinct
from its receptor binding domain [18]. In addition, leucocytes
themselves produce both inflammatory mediators and proteogly-
cans that might compete with growth factors for binding to GAGs.
In human macrophage-conditioned medium there is an inhibitor of
local vascular microenvironment. For example, rabbit EC when
treated with minimally modified LDL will express the C-X-C
chemokine GRO on their surface, which then facilitates monocyte
EC adhesion [13]. It is likely that GRO is binding to PGs in the EC
glycocalyx rather than associating directly with the cell membrane,
because GRO does not contain hydrophilic stretches that could
function as a membrane anchor region, and monocyte adhesion is
substantially reduced by prior incubation with heparin. Further,
these effects are not seen with the C-C chemokine monocyte
chemoattractant protein-1 (MCP-1). The concept that chemokine
binding to PGs is specific rather than promiscuous is also supported
by Witt & Lander, who demonstrated that IL-8 and GRO bind to
the same fractions of HSPG, whereas platelet factor-4 (PF-4) and
neutrophil-activating protein-2 (NAP-2) bind to distinct fractions.
Furthermore, they showed that differential binding is determined
by the presence of glutamic residues in the GAG binding sequence
of the chemokine [14]. The retention of chemokines at their site of
secretion by binding PG provides site-specific functional restric-
tion and may explain why soluble chemokines do not trigger
adhesion distant from their site of secretion. Indeed, there is
evidence that in some circumstances soluble chemokines can
inhibit adhesion, whereas endothelial-associated chemokines pro-
mote transendothelial migration [15].
The study of chemokine binding to PGs is hampered by
chemokine aggregation in solution and on the plasma membrane.
Luster et al. have used a novel approach to circumvent
these problems [16]. They constructed a fusion protein containing
the C-X-C chemokine interferon-inducible protein-10 (IP-10) and
alkaline phosphatase (AP). When the gene for this protein is
introduced into mammalian cells, they secrete a non-aggregating
monomeric fusion protein. They have shown that IP-10 AP and PF-
4 share the same GAG binding site and, in addition, share the
ability to inhibit angiogenesis/EC proliferation. The binding of IP-
10 to ECs is specific, saturable and competitively blocked by PF-4,
heparin and heparan sulphate. In contrast, other chemokines,
EC growth that is distinct from TGF- and TNF- [19].
In autoimmune disease GAGs may be targets for humoral
immune processes. The autoimmune antiphospholipid syndrome
is characterized by venous and arterial thromboembolism, strokes
and recurrent abortions [20], and antibodies against phospholipids
(aPLs) which disrupt normal thrombogenesis [21]. GAGs play a
vital role in the maintenence of vascular homeostasis by modulat-
ing anti-thrombin and coagulant activity on the EC surface through
interactions with anti-thrombin III (AT-III) and heparin cofactor II
[22]. It has recently been shown that a subset of aPLs with high
affinity for HS/heparin epitopes may inhibit the function of HS, so
promoting a procoagulant state [23]. These antibodies react speci-
fically with a disaccharide in the unique heparin pentasaccharide
on vascular HSPG that binds AT-III, inhibiting the formation of
thrombinAT-III complexes. In addition, other studies have shown
antibodies to HS in systemic lupus erythematosus [2426] and in
the eluate from the glomeruli of patients with renal disease [27]. A
pathogenic role for these antibodies is suggested by observations
that, in animal models, MoAbs to HS induce heavy proteinuria
[28].
In conclusion, endothelial PGs are important in the modulation
of normal immune processes and as potential targets in auto-
immune diseases. Rix and colleagues suggest that through binding
to GAGs, heparin may have a therapeutic role as a non-specific
immunosuppressant. Indeed, although currently out of vogue,
heparin has been previously used in immunosuppressive regimens
to treat aggressive immune-mediated disorders such as ulcerative
colitis and rapidly progressive glomerulonephritis [29,30]. How-
ever, before evaluating the role of anti-GAG therapies in human
disease, a clearer understanding of the range and subtleties of the
immunomodulating role of endothelial proteoglycans is required.
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