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*
Division of Biochemistry, Royal Holloway University of London, Egham, Surrey, United Kingdom;
Department of Haematology, Imperial College School of Medicine, London, United Kingdom; and
School of Pharmacy, Biomedical and Physical Sciences, University of Portsmouth, Portsmouth, Hampshire, United Kingdom
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Abstract |
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0.1 µg/ml, corresponding to 10 nM. By interpolation
with our previous findings, this indicates an affinity for heparin
greater than that of antithrombin III and comparable with that of
FGF-2, two high-affinity heparin-binding proteins. Recombinant murine
IL-12 also binds strongly to heparin. The binding of rhIL-12 to heparin
shows specificity because chondroitin sulfates A and C fail to compete,
whereas chondroitin B inhibits weakly. A highly sulfated heparan
sulfate is a strong competitor, whereas other heparan sulfates show
weak or no activity. Small heparin fragments inhibit binding, although
activity decreases with size. An octasaccharide pool derived by
cleavage of heparin with nitrous acid is a significantly stronger
inhibitor than its heparinase I-derived counterpart, further indicating
structural specificity in the interaction between rhIL-12 and heparin.
The binding of recombinant p40 to heparin appears indistinguishable
from that of the IL-12 heterodimer, implying that the heparin binding
site is largely if not solely located in this subunit. These results
show for the first time that IL-12 is a heparin-binding cytokine, a
property common to the other Th1-response-inducing cytokines, IFN-
and IL-2. Our findings strongly suggest that IL-12 will tend to be
retained close to its sites of secretion in the tissues by binding to
heparin-like glycosaminoglycans, thus favoring a paracrine role for
IL-12. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
by both resting and activated NK and T cells. Because IFN- in turn
enhances IL-12 production, a positive feedback loop exists. IL-12
induces proliferation of NK and T cells, especially in the presence of
costimulation through the B7-CD28 receptor-ligand pairing (4). It also
enhances the cytotoxic activity within these cell populations (3). An
important activity of IL-12, acting together with IFN- and IL-2, is
to drive Th cell responses toward the Th1 rather than Th2 phenotype
(3). Mice homozygous for a null mutation of the IL-12 p40 gene develop
without hematological abnormalities, but show markedly reduced IFN-
production on Ag challenge and substantially reduced delayed-type
hypersensitivity responses (5). Studies of several animal models of
infectious diseases have demonstrated a key role for the generation of
resistance via Th1 responses to parasitic protozoa such as
Leishmania major and Plasmodium cynomolgi
(6, 7, 8). Furthermore, administration of rIL-12 has also been shown to
generate potent antimetastatic and antitumor responses to several
transplantable murine tumors (9). Moreover, IL-12 is also important in
resistance to viral disease (10).
Because the cytokines IL-12, IL-2, and IFN- are soluble
proteins that are likely to diffuse rapidly following secretion, an
important question is how the inflammatory and immune responses they
stimulate remain focused at local tissue sites of infection. An
emerging mechanism favoring paracrine rather than systemic activation
is that a number of cytokines bind to sulfated polysaccharides, in
particular heparin and heparan sulfate
(HS)3. These
glycosaminoglycans are long, unbranched chains of acidic carbohydrate
occurring on the cell surface and in the extracellular matrix. The
growing list of cytokines found to be capable of binding to heparin and
HS includes those involved in the stimulation of Th1 responses by
IL-12. Thus, IFN- binds to heparin, probably via a cluster of basic
amino acid residues at its carboxyl terminus (11). More recently, it
has been proposed that interaction with HS may give rise to the
dimerisation of IFN- (12). In our laboratory, we have employed an
ELISA method, which involves the binding of a cytokine to an
immobilized heparin-BSA complex, to demonstrate that human rIL-2
(rhIL-2) binds to heparin with high affinity, Kd 0.5 µM. This interaction is specific as chondroitin
sulfates A, B, and C fail to compete, and among HS only a highly
sulfated variant was found to be an effective competitor (13). The
interaction of rhIL-2 with heparin does not appear to either increase
or decrease its biological activity in vitro, and is likely to take
place at sites on the surface of the cytokine that are distinct from
the binding sites for the IL-2R subunits (14). Overall, the binding of
these cytokines to heparin and HS is likely to retain them close to
their sites of secretion, thus serving to maintain high local
concentrations of these soluble mediators.
In the present study, we have employed our ELISA technique to demonstrate for the first time that rhIL-12 is a cytokine with high affinity for heparin. We show that this interaction with glycosaminoglycan is selective as chondroitin sulfates A and C fail to compete with the immobilized heparin complex. However, certain HS, particularly those with high sulfate densities, do interact with IL-12.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Porcine intestinal mucosal heparin (sodium salt, grade I-A), chondroitin sulfate A (from bovine trachea), chondroitin sulfate B (dermatan sulfate, from bovine mucosa), chondroitin sulfate C (from shark cartilage), fucoidan (from Fucus vesiculosus), and HS from bovine intestinal mucosa (HSBI) and from bovine kidney (HSK), were all purchased from Sigma-Aldrich (Poole, U.K.). Before use, heparin was exhaustively dialysed against initially 1 M NaCl and subsequently deionised water before freeze drying. Two HS isolated from porcine intestinal mucosa, HSA and HSE, were kindly provided by Dr. B. Mulloy (NIBSC, Hertfordshire, U.K.). HSA and HSE have Mr of 20 kDa and 8 kDa; sulfate to carboxylate ratios of 1 and 1.7; and, N-acetyl to carboxylate ratios of 0.6 and 0.2, respectively (15). The clinical low m.w. heparins, Fragmin (KabiVitrum, Stockholm, Sweden), Fraxiparine (Sanofi Chimie, Notre Dame de Bondeville, France), and Tinzaparin (Logiparin, Novo Nordisk, Gentofte, Denmark) were obtained from their respective suppliers.
Human serum albumin (99% purity) and p-nitrophenol
phosphate FAST tablets were purchased from Sigma-Aldrich. Nunc
Maxisorp ELISA plates were obtained from Life Technologies (Paisley,
Scotland).
Abs and recombinant cytokines.
rhIL-12 and and recombinant murine IL-12 (rmIL-12), as well as recombinant p40, were routinely purchased from R&D Systems (Abingdon, U.K.), although rhIL-12 was also obtained from PharMingen (San Diego, CA). Goat polyclonal Abs specific for either human or murine IL-12 were also obtained from R&D Systems. Murine anti-human IL-12 mAbs recognizing the human IL-12 p40 subunit were kindly donated by R. Carter (Imperial Cancer Research Fund, Leeds, U.K.) (16). Alkaline phosphate-coupled rabbit anti-goat and sheep anti-mouse IgG secondary Abs were purchased from Sigma-Aldrich.
Heparin-binding ELISA
A heparin-BSA complex in which heparin chains are covalently coupled via their reducing ends to the protein using sodium cyanoborohydride was synthesized as described previously (13), except that the coupling reaction mixture contained 680 mg of heparin together with 34 mg BSA. Mock-treated BSA was prepared by exposing the same concentration of BSA to sodium cyanoborohydride in the absence of heparin. In some experiments (data not shown), heparin-BSA complex (purchased from Sigma-Aldrich) was used in place of the above complex.
Heparin-binding ELISA were performed essentially as described previously (13) but with slight modification. Briefly, ELISA plate wells were coated with 100 µl 50 mM Tris-HCl buffer, pH 7.4, containing 12.7 mM EDTA and either 0.08 µg complex (measured by BSA content) or the same amount of mock-treated BSA. After washing three times with PBS containing 0.05% (v/v) Tween 20 and blocking with 2% (w/v) dried skim milk powder (Marvel, Premier Beverages, Adbaston, Staffordshire, U.K.), wells were incubated for 2 h with rIL-12 diluted in PBS. Wells were then washed three times with PBS-Tween and anti-IL-12 polyclonal Ab or mAb was added at a dilution of 1/1000 in blocking buffer. Following three further washes in PBS-Tween, the corresponding alkaline phosphatase-coupled secondary Ab was added at a dilution of 1/1000 in blocking buffer for 30 min. After five washes in PBS-Tween, 100 µl 0.2 M Tris buffer, pH 10, containing 1 mg/ml p-nitrophenol phosphate was added, and absorbances were read at 405 nm after 30- to 90-min incubation at room temperature. In some experiments, a competitive variant of the ELISA was used in which cytokine diluted in PBS was preincubated with soluble glycosaminoglycan for 5 min before the addition of 100 µl aliquots of this mixture to coated and blocked wells.
Heparin-derived oligosaccharides
Porcine intestinal mucosal heparin, 10 mg/ml, was dissolved in 250 mM sodium acetate buffer, pH 7.5, containing 2.5 mM calcium acetate. Heparinase I from Flavobacterium heparinum (Grampian Enzymes, Aberdeen, Scotland) was added at 0.07% (v/v), and the mixture was incubated at 30°C for 10 h. After boiling, the mixture was freeze dried and reconstituted in 2% (w/v) ammonium bicarbonate buffer, pH 7.6, before gel filtration on a 5 x 100-cm column of Biogel P6 equilibrated in the same buffer. The eluate was analyzed for the presence of glycosaminoglycan by a microtiter plate dimethylene blue binding assay, and the size and homogeneity of the oligosaccharide fractions was verified by PAGE, both methods being fully described elsewhere (17). Oligosaccharides were also obtained by cleavage of heparin with nitrous acid (18) as fully described elsewhere, with similar fractionation of the resulting oligosaccharide size pools.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A, dose-dependent binding to
the heparin complex was observed. This binding is detected with high
sensitivity, being readily detectable with as little as 2 ng rhIL-12.
Essentially identical results were found when rhIL-12 was obtained from
an alternative commercial source (PharMingen). By contrast, there is
negligible binding to wells coated instead with mock-treated BSA. A
strong heparin-binding response is also observed when the polyclonal
anti-IL-12 Ab is replaced with the murine anti-IL-12 mAb, 1.105
(Fig. 1B). Essentially identical dose-response curves were
obtained with the alternative anti-human IL-12 mAbs, 1.3A3 and
2.1B3 (data not shown). These findings establish that the observed
heparin binding is not an artifact of the Abs employed in the detection
of bound IL-12. In some experiments, absorbance readings taken at early
times of substrate incubation show that the binding curves were
initially quasilinear, but with increasing times they become flattened
at higher cytokine levels as the absorbances increase (Fig. 1). This
indicates that exhaustion of one or more components in the detection
system may be occurring. Thus, the shape of the curves cannot
necessarily be taken as representing saturation of the IL-12 binding
sites. rmIL-12 also shows very similar dose-dependent binding to the
immobilized heparin complex, with a strong response being readily
detectable with 1 ng of cytokine (Fig. 1C). The large
absorbances obtained in these various binding curves are in marked
contrast to similar binding experiments performed with rhIL-1
,
rhIL-1ß, and recombinant human granulocyte-macrophage CSF, in which
no or negligible binding responses were obtained (data not shown). When
the heparin-BSA complex synthesized in this laboratory was substituted
with the same amount of a commercially obtained counterpart, an
essentially identical binding curve for rhIL-12 was obtained (data not
shown).
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A, the strong binding of
rhIL-12 to the heparin complex is completely inhibited by <10 µg/ml
heparin, with an IC50 of 0.1 µg/ml. As shown in Fig. 2B, an essentially identical displacement curve is obtained
with mIL-12, giving a similar IC50 value. The potent
competition obtained with soluble heparin demonstrates that the binding
of both human and murine IL-12 to the heparin-BSA complex is due to
interaction with the immobilized heparin chains.
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A, in addition to heparin,
fucoidan, a highly sulfated fucose polymer isolated from a marine
algae, is an effective inhibitor of the binding of rhIL-12 to the
immobilized heparin complex. By contrast, neither chondroitin sulfate
A, nor chondroitin sulfate C gives significant inhibition of binding
(p = 0.05, by ANOVA with posthoc analysis on
data from three independent experiments). In contrast, chondroitin
sulfate B (dermatan sulfate) and HSBI do cause significant inhibition
(p = 0.05). However, these latter two
glycosaminoglycans are weak inhibitors, as under the conditions
employed they give inhibition in the range 10–20% compared with the
substantial inhibitions observed with heparin and fucoidan.
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B, HSBI and HSK both significantly inhibit rhIL-12 binding
(p = 0.05, by ANOVA of data from three
independent experiments), although in each case the inhibition obtained
is less than that with the same concentration of heparin. By contrast,
HSA from porcine intestinal mucosa gives no significant inhibition
(p = 0.05), whereas HSE, a more highly sulfated
preparation from the same source, is a relatively potent inhibitor of
binding.
The inhibitory activity of HSBI and HSE were investigated further by
preincubating a fixed concentration of cytokine with increasing
concentrations of these soluble glycosaminoglycans. As may be seen in
Fig. 4, HSBI is confirmed as a weak
inhibitor. At concentrations between 2 and 10 µg/ml, some 20%
inhibition of binding is observed with no clearly apparent
concentration-dependent increase. The same concentration range of
heparin gives >95% inhibition of rhIL-12 binding. The highly sulfated
HS, HSE, which in Fig. 3A gave 50% inhibition, is seen in
Fig. 4 to give 75% inhibition. Indeed, some quantitative variation in
the inhibition obtained with a particular glycosaminoglycan was found
between individual experiments. However, as may be seen in Fig. 4, this
level of inhibition is not concentration-dependent as the titration
curve attains a plateau between 2 and 10 µg/ml. Thus, these partial
inhibitors appear to be unable to provide complete inhibition of IL-12
binding to the immobilized complex even when added at concentrations
10-fold higher than 1 µg/ml, at which >95% inhibition is attained
with soluble heparin (Figs. 2 and 4). One implication of this is that
the efficacy of partial inhibitors cannot be compared by using
IC50 values because some inhibitors, for example HSBI, do
not routinely achieve 50% inhibition.
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10–17 hexose residues in length. When
used as soluble competitors at 2 µg/ml, these three preparations gave
inhibitions of binding between 75 and 85%, thus showing little or no
diminution from the 85% inhibition obtained with unfractionated,
normal heparin, 16 kDa (data not shown). Therefore, to examine the
inhibitory activity in even smaller heparin fragments, we investigated
oligosaccharides generated from heparin by digestion with heparinase I.
As may be seen in Fig. 5A,
under the conditions employed tetradecasaccharides give 90%
inhibition, a value which approaches that obtained with unfractionated
heparin in the same experiment. With each stepwise disaccharide
decrease in size there is a progressive loss of inhibition such that
decasaccharides give 50% inhibition, whereas with tetrasaccharides
inhibition is essentially undetectable. A similar study using
heparin-derived oligosaccharides obtained by depolymerisation with
nitrous acid also showed that small fragments compete for the binding
of rhIL-12. As may be seen in Fig. 5B, the
tetradecasaccharides obtained in this way, like those produced with
heparinase I, give almost complete inhibition of binding under the
conditions employed. Again, with decreasing oligosaccharide size there
is a stepwise decrease in activity, with the tetrasaccharides and
hexasaccharides being weak inhibitors. However, with the nitrous acid
fragments, the decrease in activity between the dodecasaccharides and
the octasaccharides is less marked compared with that seen with the
heparinase I products. Thus, the nitrous acid-derived octasaccharides
give rise to >50% inhibition of binding, whereas its enzymically
produced counterpart does not. The greater activity of the nitrous
acid-cleaved fragment was examined by titration of the inhibitory
activity of both octasaccharides over the range 0–10 µg/ml. With the
nitrous acid-derived and heparinase-derived octasaccharides, the
plateaux of maximal inhibition were at 55% and 25%, respectively
(data not shown), thus confirming that the former provides around twice
the inhibition of binding compared with the latter.
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A, p40, like intact IL-12,
shows strong binding to the immobilized heparin complex, with as little
as 5 ng/well giving rise to a substantial absorbance that increases
further at high IL-12 loadings. Similar binding curves were also
obtained using polyclonal anti-IL-12 Abs, as well as other
anti-IL-12 mAbs (data not shown). As may be seen in Fig. 6B, the binding of p40 is readily displaceable on
preincubation with soluble heparin with an IC50 of 0.1
µg/ml. Therefore, taken overall the heparin-binding properties of p40
closely resemble those of the IL-12 heterodimer. The lack of
availability of both recombinant p35 subunit preparations and
p35-specific mAbs precludes the study of the heparin-binding properties
of the smaller IL-12 subunit in an isolated state.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our study shows that relatively small heparin-derived oligosaccharides compete for binding to the intact heparin chains. Substantial inhibition of binding, exceeding 50% in the case of nitrous acid-derived oligosaccharides, is obtained with fragments as small as octamers. Given the considerable sequence heterogeneity of heparin chains, each oligosaccharide size pool will contain a large range of different sequences, the diversity of which will increase with size. The two heparin cleavage methods employed here have different linkage specificities, with heparinase 1 selectively cleaving between N-sulfated glucoamines and 2-O-sulfated iduronates (22), whereas nitrous acid is less selective. Thus the generation of less active octasaccharides by the enzymic route compared with nitrous acid suggests a role for N-sulfated glucoamine-2-O-sulfated iduronate disaccharides in the binding of IL-12. Such disaccharides have already been implicated in FGF-2 binding (23, 24, 25). This disaccharide is relatively abundant in heparin, but less common in HS, particularly where this has a low overall sulfate content (26). Thus, our finding that the competitive activity is in the order heparin > high sulfated HS > low sulfated HS is entirely consistent with a role for sequences containing this disaccharide.
The best characterized heparin-protein interaction, the high-affinity
binding of heparin to antithrombin III, involves a defined
pentasaccharide sequence (27). The minimal sequence for the cytokine
FGF-2 binding is less certain as different studies have implicated
oligosaccharides varying from hexasaccharide (23, 24) to decasaccharide
(25). However, in the case of IFN-, the HS-binding sequence appears
to be 10 kDa, corresponding to over 40 hexose residues. This large
fragment is envisaged as spanning across the two identical subunits in
the homodimeric IFN- molecule, interacting with basic residues at
the two C termini (12). Similarly the tetrameric chemokine, platelet
factor 4, is bound by a 42-residue HS fragment that is envisaged to
wrap around the molecule interacting with all four polypeptide subunits
(28). Taken overall, our observation that comparatively small
oligosaccharides are effective competitors for heparin binding suggest
that only a single binding site is involved in IL-12 binding.
Advancement of this hypothesis requires further study, including
definition of the minimal binding sequence.
We show here that recombinant p40 subunits possess heparin-binding properties indistinguishable from those of the heterodimeric IL-12 molecule. Lack of availability of appropriate recombinant reagents prevents examination in the same way of the possible interaction of p35 with heparin. However, our current data indicates that the heparin binding of IL-12 is at least largely localized in the larger subunit. Studies of human/mouse hybrid combinations of the p35 and p40 subunits implicate p35 as largely responsible for receptor interactions (5). This finding reflects the fact that this smaller subunit displays a low homology with IL-6 and granulocyte CSF (29), whereas p40 has extensive albeit weak homology with the receptors for IL-6 and ciliary neurotrophic factor (5, 30). It is also significant that IL-12 secretion appears to be associated with excess p40 secretion and that the excess p40 acting either singly or as homodimers may function as a receptor antagonist of heterodimeric IL-12 (3). Thus, our present observations suggest heparin binds to the relatively accessory subunit rather than the smaller subunit, which appears to be the more important in signal transduction.
Given the strongly acidic nature of heparin and HS, the basic amino
acids arginine and lysine are major determinants of their binding sites
on polypeptides. It is quite possible for secondary structural folding
of a polypeptide to bring into proximity basic residues that in the
primary sequence are well separated from each other, so as to
constitute discontinuous heparin binding sites (31). However, it is
notable that human p40 possesses near its C terminus a cluster of six
basic residues within a nine-amino acid sequence, as may be seen in
Fig. 7. This unusually dense basic
sequence is a prime candidate for a heparin binding site. We have found
that murine IL-12 also binds heparin, and interestingly although the
C-terminal region of murine p40 has little overall homology with that
of its human counterpart, it too has a sequence cluster of six basic
residues (Fig. 7). These clusters are comparable to the C-terminal
proximal clusters of 4–5 basic residues that give rise to the
heparin-binding properties of IFN- (11), IL-8 (33), and other
CXC chemokine family members (34).
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Therefore, we establish here that IL-12 binds with high affinity to
heparin/HS glycosaminoglycan. This interaction is likely to retain
IL-12 close to its tissue sites of secretion, establishing local high
concentrations. Heparin is a constituent of the cytoplasmic granules of
mast cells, which as a result of mast cell degranulation at tissues
sites of inflammation will become a component of the extracellular
milieu (37). IL-12, in cooperation with IFN- and IL-2, has a major
role in establishing immune responses of a Th1 type. Therefore, it is
of considerable interest that both IFN- and IL-2 have previously
been shown to bind to the same glycosaminoglycans (11, 12, 13, 14). Thus, this
IL activation of the cell-mediated immunity may well be exerted within
local tissue compartments by the combined activity of these
heparin-retained cytokines. It has recently been shown that splenic
dendritic cells respond rapidly to microbial challenge by secreting
large amounts of IL-12 and migrating into T cell areas of the white
pulp (38). The resulting close proximity of these dendritic cells to
the T cell population they stimulate clearly exemplifies the short
distances over which IL-12 functions physiologically. Moreover,
systemically administered rIL-12 is associated with several toxic side
effects (39). If endogenously secreted IL-12 is indeed localized in
tissue compartments by retention on heparin/HS glycosaminoglycan, this
would explain why the systemic route of administration is
inappropriate.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christopher C. Rider, Division of Biochemistry, Royal Holloway University of London, Egham Hill, Egham, Surrey TW20 0EX, U.K.
3 Abbreviations used in this paper: HS, heparan sulfate; h, human; m, mouse; HSBI, HS from bovine intestinal mucosa; HSK, HS from bovine kidney.
Received for publication May 15, 1998. Accepted for publication September 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and type 1 cytokine responses. Immunity 4:471.[Medline]
binds to heparan sulfate by a cluster of amino acids located in the C-terminal part of the molecule. FEBS Letts. 280:152.[Medline]
-binding domain in heparan sulphate. Biochem. J. 310:497.
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) or mock-treated BSA (
).
A, Binding of rhIL-12 was detected with goat polyclonal
anti-IL-12, and absorbances were read after 30 min incubation with
substrate. The curves shown are typical of six independent experiments.
B, Binding of rhIL-12 was detected with mAb 1.105. The
curves are typical of two independent experiments, and absorbances were
read after 1 h. C, Binding of rmIL-12 was detected
with polyclonal Ab. The curves are typical of two independent
experiments and were obtained after 30 min incubation. Throughout blank
absorbance values, typically 0.1–0.15, obtained with wells containing
only substrate have been subtracted from the values shown. Error bars
indicate ± SEM of the quadruplicates, where this is larger than
the symbol size.
