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,
Departments of
*
Surgery,
Immunology, and
Pediatrics, Mayo Clinic, Rochester, MN 55905;
§
Department of Surgery, Duke University Medical Center, Durham, NC 27710; and
¶
Department of Surgery, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1. How dendritic cells might be
activated in the absence of exogenous stimuli, or without the immediate
presence of activated leukocytes, as might occur in immunity to tumor
cells or transplants, is unknown. We postulated that heparan sulfate,
an acidic, biologically active polysaccharide associated with cell
membranes and extracellular matrices, which is rapidly released under
conditions of inflammation and tissue damage, might provide such a
stimulus. Incubation of immature murine dendritic cells with heparan
sulfate induced phenotypic maturation evidenced by up-regulation of
I-A, CD40, CD54 (ICAM-1), CD80 (B7-1), and CD86 (B7-2). Dendritic cells
exposed to heparan sulfate exhibited a markedly lowered rate of Ag
uptake and increased allostimulatory capacity. Stimulation of dendritic
cells with heparan sulfate induced release of TNF-, IL-1ß, and
IL-6, although the maturation of dendritic cells was independent of
these cytokines. These results suggest that soluble heparan sulfate
chains, as products of the degradation of heparan sulfate proteoglycan,
might induce maturation of dendritic cells without exogenous stimuli,
thus contributing to the generation and maintenance of primary immune
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dendritic cells can be induced into mature immunostimulatory APC by
microbial components (3), such as LPS (4) and
dsRNA (5); by cytokines, such as TNF-, IL-1, and type I
IFN (4, 6, 7, 8); and by ligation of CD40 (9).
Maturation of dendritic cells can also be induced by ligation of Fc
receptors (10), CD43 (11), and CD44
(12), by cell adhesion (13, 14), and by
prostaglandins (15, 16). These stimuli require the local
presence of microorganisms or activated leukocytes. How primary immune
reactions, as might be seen in response to viral and tumor Ags and to
transplants, are induced in the absence of these inflammatory mediators
is unknown. Gallucci et al. (17) call these mediators of
dendritic cell activation, natural adjuvants; however, the identity of
such natural adjuvants is, likewise, unknown. We postulated
that one component of normal tissues, which might mediate this effect,
is heparan sulfate.
Heparan sulfate is a linear copolymer, comprised of repeating hexuronic acid and hexosamine residues, which is found ubiquitously distributed on cell surfaces and extracellular matrices. As a component of proteoglycans, the heparan sulfate polymers are covalently attached to a protein core (18). Structural modifications of the saccharide residues, especially sulfation, confer manifold biological activities, including regulation of cell adhesion, proliferation, development, anticoagulant, and chemical mediator functions (18, 19, 20, 21). Injury of tissues (22) or exposure of endothelial cells and, perhaps, other cells to activated complement or to neutrophils or platelets causes rapid cleavage and shedding of heparan sulfate proteoglycans and glycosaminoglycan fragments (23, 24, 25, 26). The release of heparan sulfate is postulated to be mediated by proteolytic cleavage of the protein core or by endoglycolytic cleavage of the heparan sulfate chains (25, 27, 28, 29).
We and others have proposed that the release of heparan sulfate
proteoglycans may play an important role in the regulation and
manifestation of immune responses in the local inflammatory sites
(21, 30). Consistent with this hypothesis, we previously
found that soluble heparan sulfate delivers signals to macrophages,
including activation of tyrosine kinase and protein kinase C, calcium
influx, elevation of inositol phosphate, and NF-
B translocation
(31). These events cause murine macrophages to release
IL-1, IL-6, and PGE2 (32), and to
up-regulate MHC class II and CD86 (33), profoundly
increasing the ability of the macrophages to modify immune
responses.
Because the conditions associated with maturation and migration of dendritic cells might be accompanied by degradation of heparan sulfate proteoglycans in peripheral tissues, we hypothesized that soluble heparan sulfate might be one factor promoting the maturation of dendritic cells. To test this idea, we stimulated murine bone marrow-derived dendritic cells with soluble heparan sulfate, without exogenous cytokines, analyzing the phenotypic and functional changes that ensued. We report, in this study, that interaction with soluble heparan sulfate induces maturation of dendritic cells, suggesting that release of heparan sulfate proteoglycans may provide an endogenous stimulus to induce cellular immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FITC-conjugated mAbs, HL3 (anti-CD11c), HM40-3
(anti-CD40), RA3-6B2 (anti-CD45R/B220), 3E2 (anti-CD54:
ICAM-1), 16-10A1 (anti-CD80: B7-1), GL1 (anti-CD86: B7-2),
RB6-8C5 (anti-Ly-6G: Gr-1), G235-2356 (hamster IgG isotype
standard), and R35-95 (rat IgG isotype standard); biotin-conjugated
Abs, AF6-88.5 (anti-H-2Kb), AF6-120.1
(anti-I-Ab), PE-conjugated streptavidin, and
2.4G2 (anti-CD16/CD32); neutralizing Abs, 35F5 (anti-CD121a:
IL-1R type I/p80), MP5-20F3 (anti-IL-6), G281-2626
(anti-TNF-), and R3-34 (isotype control for rat IgG1);
recombinant murine TNF- and IL-6 were purchased from PharMingen (San
Diego, CA). FITC-conjugated anti-macrophage F4/80 Ag mAb was from
Caltag (Burlingame, CA). Neutralizing Abs contained 
0.01 ng
endotoxin/µg protein. Heparan sulfate (from bovine kidney),
Limulus amebocyte assay (E-TOXATE), and LPS from
Escherichia coli were from Sigma (St. Louis, MO). An
endotoxin filter (END-X) and endotoxin removal resin (END-X B15) were
from Associates of Cape Cod (Woods Hole, MA). Cytokine ELISA kits for
murine IL-1ß, IL-6, and TNF-, and murine rIL-1ß were purchased
from R&D Systems (Minneapolis, MN).
Isolation and culture of dendritic cells
Dendritic cells were generated from murine bone marrow cells, as described by Inaba et al. (34, 35), with minor modifications. Briefly, bone marrow was flushed from the long bones of C57BL/6 mice (Charles River, Wilmington, MA) and depleted of red cells with ammonium chloride. Cells were plated in six-well culture plates (106 cells/ml, 3 ml/well) in RPMI 1640 supplemented with 5% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 x 10-5 M 2-ME, 10 mM HEPES (pH 7.4) (Life Technologies, Gaithersburg, MD), and 3.3 ng/ml of murine rGM-CSF at 37°C, 5% CO2. At day 3 of culture, floating cells were gently removed and fresh medium was added. At day 6 or day 7 of culture, nonadherent cells and loosely adherent proliferating dendritic cell aggregates were harvested for analysis or stimulation, or, in some experiments, replated in 60-mm petri dishes (106 cells/ml, 5 ml/dish). At day 10 of culture, nonadherent cells (dendritic cells) were removed for analysis. Less than 1% of cells were B220+, and less than 10% were Gr-1+ analyzed by flow cytometry (not shown). Dendritic cells were prepared by cytospin (Shandon, Pittsburgh, PA) for morphological analysis with Wright-Giemsa stain (EM Diagnostic Systems, Gibbstown, NJ).
In certain experiments, CD11c+ dendritic cells were isolated from cultured bone marrow cells using anti-CD11c (N418) microbeads and a magnetic cell sorting system (Vario MACS; Miltenyi Biotec, Auburn, CA). Purity of the selected cell fraction was >92%.
Stimulation of dendritic cells by heparan sulfate
Heparan sulfate dissolved in distilled water, or distilled water
alone, as control, was added to cultures of isolated dendritic cells in
six-well plates (106 cells/ml, 3 ml/well).
Heparan sulfate was treated with LPS-binding protein, conjugated to a
filter or resin to remove endotoxin (36). In certain
experiments, heparan sulfate was pretreated with
HNO2 at pH 1.5 to depolymerize the
glycosaminoglycan chains (37). Neutralizing Abs against
TNF-, IL-1R, and IL-6 were used at 
10-fold the concentrations
needed for 50% inhibition, as reported in the manufacturer’s
instructions.
Allogeneic MLR
Splenocytes, isolated from female BALB/c mice at 5–8 wk of age (Charles River), were passed over nylon wool columns to enrich T cells. Nylon wool nonadherent splenocytes (4 x 105/well) were cocultured with irradiated (15 Gy) dendritic cells isolated from C57BL/6 mice, as described above. Mixed cultures were conducted in RPMI 1640 supplemented with 1% mouse serum (from BALB/c mice), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 x 10-5 M 2-ME, and 10 mM HEPES (pH 7.4) in 96-well round-bottom plates at 37°C, 5% CO2 in air. Cell proliferation was estimated based on uptake of [3H]thymidine (ICN, Irvine, CA). For this purpose, cells were pulsed with 5 µCi/ml of [3H]thymidine for 18 h, with the radioactivity of harvested cells, then measured by a liquid scintillation counter (Wallac Oy, Turku, Finland).
Flow cytometric analysis
Flow cytometric analysis was performed, as described by Kodaira et al. (38). Briefly, 5 x 105 cells were incubated in staining buffer (PBS with 2% FBS and 0.1% sodium azide) containing anti-CD16/CD32 Ab (PharMingen) to block nonspecific binding of Igs for 5 min on ice. Cells were then stained, as indicated, and analyzed by FACScan, using CellQuest software (Becton Dickinson, San Jose, CA). FACStar cell sorter was used for cell sorting (Becton Dickinson).
Quantitation of Ag uptake
Endocytosis was quantitated, as described by Lutz et al. (39) and Sallusto et al. (4). Briefly, 2 x 105 cells were equilibrated at 37°C or 0°C for 10 min and then pulsed with fluorescein-conjugated dextran (40,000 m.w.; Molecular Probes, Eugene, OR) at a concentration of 1 mg/ml. After different times in incubation, at 37°C or 0°C, cold staining buffer was added to stop the reaction. Cells were washed three times and stained with biotin-conjugated anti-I-Ab Abs and streptavidin-PE, then analyzed by FACScan. Nonspecific binding of dextran to dendritic cells, determined by incubation of dendritic cells with fluorescein-conjugated dextran at 0°C, was subtracted. The medium used in the culture, to stimulate dendritic cells with heparan sulfate, was supplemented with GM-CSF, because the ability of dendritic cells to capture Ag is lost if dendritic cells are cultured without GM-CSF (data not shown) (4).
Cytokine assay
Culture supernatants were analyzed by ELISA. OD at 450 nm of
duplicate samples was determined and corrected by a microplate reader,
with readings at 570 nm (PowerWavex with KC4
software; Bio-Tek, Winooski, VT). The minimum detection levels were as
follows: IL-1ß, 3 pg/ml; IL-6, 3.1 pg/ml; and TNF-, 5.1 pg/ml.
Statistics
The Mann-Whitney U test was used for statistical analysis. A p value of less than 0.005 was considered to indicate statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After 6 days of culture with GM-CSF, the nonadherent fraction of
murine bone marrow cells consisted mainly (70%) of immature
dendritic cells with a phenotype of CD11c+, MHC
class IIintermediate,
CD40low, CD54low (ICAM-1),
and CD86low (B7-2) (Fig. 1
), consistent with previous reports
(34, 40). After 10 days of culture with GM-CSF, 80–90%
of cells exhibited a relatively mature dendritic cell phenotype based
on expression of CD11c, I-A, CD40, CD54, CD80, and CD86 (Fig. 1). While
expression of MHC class II, CD40, and CD86 can be detected at day 10,
the expression of these molecules is well below maximum levels (seen in
stimulated dendritic cells, as illustrated in Fig. 2).
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To test whether heparan sulfate influences the maturation of
dendritic cells, immature dendritic cells from day 6 murine bone marrow
cultures were incubated for 48 h, with heparan sulfate without
exogenous cytokines. Analysis of the heparan sulfate-treated cells
revealed up-regulation of I-A on dendritic cells. Increased expression
of CD40 and CD86 (B7-2), and, to a lesser extent, up-regulation of CD54
(ICAM-1) and CD80 (B7-1), were detected on the
I-A+ cells (Fig. 2
a). Not only was the
intensity of I-A, CD40, and CD86 increased, but the fraction of cells
expressing these molecules was increased by heparan sulfate in a
dose-dependent fashion. Increase of
I-A+/CD40+ and
I-A+/CD86+ cell fractions
and the levels of expression of the costimulatory molecules induced by
heparan sulfate was comparable with changes induced by LPS (Fig. 2, a and b). More than 98% of sorted
I-A+/CD86+ cells exhibited
morphological characteristics of dendritic cells, such as extended cell
processes and large size (not shown). Although up-regulation of I-A and
slight increase of CD54 were observed in control cells, increase in
expression of CD40 and CD86 was not observed without stimulation of
dendritic cells with heparan sulfate or LPS. The reason for
up-regulation of I-A and CD54 is unclear; however, the control cells
may be partially stimulated by mechanical manipulation during transfer
between culture plates (17). More than 98% of sorted
I-A+/CD86+ cells exhibited
morphological characteristics of dendritic cells, such as extended
cell processes and large size (not shown). The maturation of dendritic
cells was not the result of lipid or protein contaminants of heparan
sulfate because: 1) only a minimal level of endotoxin was detected by
Limulus amebocyte assay in heparan sulfate solution; 2)
absorbing the solution with LPS-binding protein to remove endotoxin did
not affect the maturation; 3) the same results were obtained using
heparan sulfate solution, which had been boiled for 10 min to denature
any contaminating proteins; and 4) up-regulation of CD40 and CD86 did
not occur if heparan sulfate was subjected to deaminative cleavage with
HNO2. In contrast, up-regulation of CD40 and CD86
was observed in dendritic cells stimulated with LPS that had been
treated with HNO2 (data not shown).
Maturation of dendritic cells exposed to heparan sulfate could reflect
the direct action of heparan sulfate on dendritic cells or a response
of dendritic cells to cytokines released by other cells. For example,
we previously showed that macrophages, stimulated with heparan sulfate,
release IL-1 and IL-6 (31). To rule out the possibility
that maturation of dendritic cells is induced by cytokines released
from macrophages or other cells, the responses of highly purified
dendritic cells were tested. CD11c+ dendritic
cells (>92%) also up-regulated CD40, CD54, CD80, and CD86 after
stimulation with heparan sulfate for 48 h (Table I). The same mature phenotype was
observed if immature dendritic cells were stimulated with heparan
sulfate in GM-CSF-supplemented medium, indicating that exogenous GM-CSF
did not alter the effects of heparan sulfate (not shown). Therefore,
cytokines released from cells other than dendritic cells are not
necessary for the maturation of dendritic cells induced by heparan
sulfate.
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We next asked whether stimulation of dendritic cells with heparan
sulfate changes the ability of dendritic cells to capture Ag
quantitated based on uptake of fluorescein-conjugated dextran
(41, 42). As shown in Fig. 3, dendritic cells from day 6 bone marrow
cultures, treated for 48 h with heparan sulfate, had remarkably
low uptake of Ag, compared with untreated, immature dendritic cells
(bone marrow culture at day 6) or dendritic cells treated without
heparan sulfate. These data suggest that heparan sulfate induced the
mature metabolic state of dendritic cells.
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To test whether heparan sulfate induces maturation of dendritic
cells to fully functional APC, dendritic cells from day 6 cultures that
had been incubated with heparan sulfate for 48 h were tested for
their capacity to stimulate allogeneic T cells. As Fig. 4 shows, heparan sulfate-treated
dendritic cells stimulated proliferative responses more effectively
than control dendritic cells, and as well as LPS-treated cells did.
Augmentation of proliferative responses was most evident under
suboptimal conditions in which the number of stimulator cells was
limited. The addition of GM-CSF to heparan sulfate further augmented
proliferative responses induced by dendritic cells under suboptimal
conditions (not shown). Viability of the stimulator cells incubated
with and without heparan sulfate was 95.2 ± 0.9% and 90.2
± 2.1% (mean ± SE of three experiments), respectively,
suggesting that the effects of heparan sulfate were not due to
differences in viability of dendritic cells.
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We next analyzed whether or not heparan sulfate might induce
release of cytokines by dendritic cells and whether or not such
cytokines might, in turn, mediate the maturation of dendritic cells.
Significant amounts of TNF-, IL-1ß, and IL-6 were detected in the
culture supernatant of purified CD11c+ dendritic
cells stimulated with heparan sulfate for 48 h (Fig. 5). However, as seen from results in
Table I, the up-regulation of CD40 and CD86 after stimulation with
heparan sulfate was not blocked by neutralizing Abs against TNF-,
IL-1R type I, or IL-6, nor did various combinations of
neutralizing Abs prevent phenotypic maturation (not shown). The
neutralizing activities of anti-TNF- and anti-IL-1R Abs at
the indicated concentrations were confirmed in the same model systems
as shown in Table I. The maturation of dendritic cells induced by
TNF- (10–20 ng/ml) or IL-1ß (0.2–10 ng/ml) was completely
blocked by the neutralizing Abs, but not by isotype control. The
presence of IL-6 alone, at the concentration shown in Fig. 5 (40
ng/ml), did not induce the up-regulation of CD40 and CD86 (not shown).
Furthermore, the up-regulation of CD40 and CD86 induced by a mixture of
TNF-, IL-1ß, and IL-6 was abrogated at 92% by a simultaneous
presence of the three neutralizing Abs. Therefore, TNF-, IL-1ß,
and IL-6, released from dendritic cells, do not directly contribute to
maturation of dendritic cells induced by heparan sulfate, suggesting
that signals, provided by heparan sulfate itself, or the other factors
induced by heparan sulfate, cause maturation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The major requirement for the effects of heparan sulfate on dendritic cells may be solubilization of heparan sulfate proteoglycans from normal tissues. Solubilization of heparan sulfate might be brought about by enzymatic cleavage of the protein core, the glycosaminoglycan chains, or, in the case of glypican, the lipid anchor (25, 27, 28, 29). For example, in injured or inflamed tissue, heparanase released from platelets and stabilized by acidic pH could mediate cleavage of heparanase sulfate (25, 30). The activation of complement to induce ischemia or tissue damage might cause the activation of proteases that cleave heparan sulfate proteoglycans from vascular endothelium (23). It should be noted that cleavage of heparan sulfate following activation of complement is nearly immediate, and the metabolites can be assumed to be the products of normal tissues. Because heparanase is also produced by some tumors (27, 43, 44), heparan sulfate fragments might be released in the neighborhood of invasive or metastasizing tumors. It has been reported that tumor cells undergoing necrosis provide signals for maturation of dendritic cells (17, 45), although detailed mechanisms on the induction of maturation are not completely clarified. Heparan sulfate, released from necrotic tumor, might well be one of the maturation factors that initiates tumor immunity through dendritic cell maturation.
Although it is still unclear how heparan sulfate provides signals to
APCs, direct binding of heparan sulfate to cell surface receptors is
one possibility (31). We have reported that signaling via
GPI-linked receptor was at least partially involved in activation of
murine peritoneal macrophages by heparan sulfate, leading to activation
of protein kinase C and NF-B translocation (31).
Peptidoglycan and LPS signal cells through a toll-like receptor that has been reported as a 70-kDa protein on the surface of mouse lymphocytes (46, 47). This receptor may also be shared by heparan sulfate. These reports suggest the possibility of receptor-mediated activation of APCs by heparan sulfate. On the other hand, heparan sulfate might also modify stimulation of dendritic cells by growth factors, chemokines, or cytokines. Binding of heparan sulfate to some growth factors, such as fibroblast growth factors, induces structural modifications (48) or protects the factors from degradation by proteases (22). Heparan sulfate might also provide a bridge between components of the extracellular matrix and components of cell surfaces (49), leading to induction of maturation of dendritic cells (13, 14).
The metabolism of heparan sulfate proteoglycan and action of soluble
fragments on dendritic cells might also contribute to chronic immune
reactions. For example, in rheumatoid arthritis, autoreactive T cells
are induced and maintained by local dendritic cells, with this ongoing
interaction possibly leading to formation of local lymphoid tissue
(50). Because rheumatoid synovium contains elevated levels
of cleaved glycosaminoglycans (51, 52), it is not unlikely
that heparan sulfate stimulates dendritic cells to present self Ags to
autoreactive T cells, perhaps under control of endogenous TNF-
(53), and that cytokines produced by the stimulated
dendritic cells contribute to local inflammation and tissue injury.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeffrey L. Platt, Departments of Surgery, Immunology, and Pediatrics, Mayo Clinic, Medical Sciences Building, 2-66, 200 1st Street, SW, Rochester, MN 55905.
Received for publication November 29, 1999. Accepted for publication May 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Exp. Med. 179:1109. receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class-I restricted antigen presentation after immune complex internalization. J. Exp. Med. 189:371. cooperate to activate human dendritic cells: synergistic activation of interleukin 12 production. J. Exp. Med. 186:1603.This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and
I-A+/CD40+ cells (
) in the number of total
harvested cells after stimulation, with various concentrations of
heparan sulfate or LPS, were analyzed. The bars show the mean ±
SD of two independent experiments.
) or
without (
) heparan sulfate, and bone marrow-derived dendritic cells
harvested at day 6 (
) were pulsed with
fluorescein-dextran (40,000 m.w., 1 mg/ml) and analyzed by FACS, as
described in Materials and Methods. Data shown are
representative of three experiments.
), for 2 days,
without exogenous GM-CSF. The treated dendritic cells were harvested
and washed extensively to remove heparan sulfate or LPS. MLR was
conducted for 4 days, as described in Materials and
Methods. Nonadherent cells in the bone marrow cultures at day 6
were also used for MLR as stimulators before treatment (