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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 223-229
Differential Effects of Chondroitin Sulfates A and B on Monocyte and
B-Cell Activation: Evidence for B-Cell Activation Via a CD44-Dependent
Pathway
By
Jacob Rachmilewitz and
Mark L. Tykocinski
From the Department of Pathology, Case Western Reserve University,
Cleveland, OH.
 |
ABSTRACT |
At inflammatory sites, proteoglycans are both secreted by activated
mononuclear leukocytes and released as a consequence of extracellular
matrix degradation. Chondroitin 4-sulfate proteoglycans constitute
the predominant ones produced by activated human monocytes/macrophages. In this study, we show that two chondroitin 4-sulfate forms, CSA and
CSB, can activate distinct peripheral blood mononuclear cell types.
Whereas CSA activates monocytes (to secrete monokines), CSB activates
B-cells (to proliferate). In contrast, the chondroitin 6-sulfate CSC
and heparin do not exert these functional effects. We further show that
CD44 monoclonal antibodies block CSB-induced B-cell proliferation.
These findings point to glycosaminoglycans, and specifically
chondroitin 4-sulfates, as a novel class of immunological mediators at
inflammatory sites. Furthermore, the data link CD44 to B-cell
activation, paralleling the established roles of CD44 in T-cell and
monocyte activation.
| |
INTRODUCTION |
PROTEOGLYCANS (PGs) are macromolecules
composed of a central core protein to which one or more
glycosaminoglycan (GAG) chains are covalently attached.1
GAGs are large carbohydrates that are composed of repeating
disaccharide units and exist in four forms: heparan sulfate (HS) and
heparin, chondroitin sulfate (CS, mainly CSA and CSC, and dermatan
sulfate, CSB), keratan sulfate, and hyaluronic acid (HA). The first
three occur predominantly as protein-bound GAGs and contain sulfate; HA
is made as a free GAG and lacks sulfate. In the case of the chondroitin
forms, CSA and CSB are structurally related, since both are 4-sulfated,
whereas CSC is 6-sulfated.
Stimulated monocytes and macrophages secrete a diverse set of mediators
that influence cellular immune functions and inflammation. These
mediators include pro- and anti-inflammatory cytokines, prostaglandins,
leukotrienes, and reactive oxygen metabolites.2 Soluble PGs
constitute another class of molecules produced by human
monocytes3,4 and macrophages.5 Chondroitin
sulfate proteoglycans (CSPG) have been reported to be the predominant proteoglycan produced by cells of the monocyte/macrophage lineage, and
80% to 90% of this CSPG is chondroitin 4-sulfate (ie, CSA or
CSB).5,6
Although the production of CSPGs by monocyte/macrophages during immune
activation has been well documented, information pertaining to their
functional significance is sparse. CS-modified invariant chain (Ii)
associated with major histocompatibility complex (MHC) class II has
been shown to enhance stimulation of T-cell responses through
interactions with CD44.7 Serglycin, a CSPG secreted by
hematopoietic cells,5 activates cytotoxic T-cell
clones.8 Chondroitin 4-sulfate released by stimulated
polymorphonuclear leukocytes during phagocytosis stimulates their
chemotaxis.3,9-12
These disparate studies focused narrowly upon the effects of CSPGs/CSs
on isolated immune cell populations. In the present study, we have
instead monitored the effects of GAGs, and in particular CSs, on the
mixed cell populations present within human peripheral blood
mononuclear cell (PBMC) preparations. In this way, we have sought to
search in a more open-ended way for effects on different immune cell
subpopulations, using a more complex cellular experimental milieu where
critical intercellular contacts can take place. What have emerged from
these analyses are new insights into the immunomodulatory potential of
the chondroitin 4-sulfate forms, with differing properties associated
with CSA versus CSB. Whereas CSA is directed toward monocytic cells,
CSB exerts its activating effects on B cells. These findings provide
the first immunoregulatory links between CSs and both the monocytic and
B-cell compartments. Moreover, the apparent role of CD44 in mediating
the CSB effect on human B cells provides a new functional niche for
CD44 in immunoregulation that goes beyond its documented effects on
monocytes and T cells.
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MATERIALS AND METHODS |
Chemicals and antibodies.
Chondroitin sulfates A (bovine trachea), B (pocrine intestinal mucosa),
and C (shark cartilage), heparin (pocrine intestinal mucosa),
hyaluronic acid (human umbilical cord), and chondroitinase AC were
obtained commercially (Sigma Chemical Co, St Louis, MO). Phycoerythrin-conjugated CD19 (clone 89B, IgG1) and control monoclonal antibody (MoAb) MsIgG1 were purchased from Coulter Clone (Miami, FL).
Monoclonal mouse anti-human CD44 (clone A3D8, IgG1) was obtained from
Harlan (Sussex, UK). The monoclonal and polyclonal anti-interleukin-1 (anti-IL-1) antibodies13 were kindly provided by Dr M.A.
Friedlander (Case Western Reserve University). Recombinant human
transforming growth factor- 1 (rTGF1)
was purchased from R&D Systems (Minneapolis, MN).
Cell culture.
PBMC were separated from heparinized venous blood of healthy donors by
density gradient centrifugation over Ficoll-paque
gradients and maintained as described.14 The U937 cell line
was purchased from the American Type Cell Culture Collection
(Rockville, MD) and was maintained in the above medium.
Cytokine production.
GAGs were added to PBMC cultures at the indicated concentrations.
Cultures were incubated for either 24 or 48 hours, and conditioned media were then obtained and stored at  20°C. IL-1 was
measured in 24-hour conditioned media by a sandwich enzyme-linked
immunosorbent assay (ELISA) as described previously.13
Proliferation assay.
PBMC (1 × 105 cells in 0.2 mL per well)
were cultured in 96-well plates (Falcon, Lincoln Park, NJ) in
triplicate. After stimulation, cultures were pulsed for the last 16 hours with 0.5 µCi per well [3H]-methyl-thymidine (New
England Nuclear, Boston, MA). Cells were collected with a Harvester 96 cell harvester (Tomtec, Orange, CT), and the filters were dried and
counted with a 1205 Betaplate counter (Wallac, Turku, Finland).
Immunofluorescence and flow cytometry.
PBMC were stained with the phycoerythrin-conjugated CD19 MoAb, or the
appropriate phycoerythrin-conjugated isotype-matched control MoAb, as
described.15 Immunostained cells were analyzed on a FACScan
flow cytometer (Becton Dickinson, San Jose, CA) using Lysis II
software.
Northern blot analysis.
Total cellular RNA isolation and Northern blot hybridization were
performed as previously described16 using an IL-1 probe comprising a 530-bp Nde I-BamHI cDNA fragment
corresponding to amino acids 1-139 of the IL-1 precursor.
Hybridization signals were quantitated by phosphoimaging using
InstantImager (Packard, Meriden, CT).
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RESULTS |
Induction of IL-1 secretion by CSA.
Given the importance of IL-1 in the initiation and modulation of
immune responses, we focused initially upon this monokine. Previous
studies have documented the capacity of HA to induce IL-1 and other
monokine secretions.17,18 We surveyed the capacity of five
different GAGs (CSA, CSB, CSC, heparin, and HA) to induce IL-1
secretion. Both HA and CSA reproducibly induced IL-1 secretion by
PBMC. The HA-induced IL-1 secretion was evident at concentrations as
low as 1 µg/mL (data not shown), consistent with the findings of Hiro
et al.17 CSA, on the other hand, induced detectable levels
of IL-1 in PBMC only at substantially higher concentrations (100 µg/mL to 1 mg/mL) (Fig 1A). After
phytohemagglutinin (PHA) induction, PBMC continued to show a
dose-dependent increase in IL-1 secretion over a CSA concentration
range of 10 µg/mL to 1 mg/mL (Fig 1B).
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| Fig 1.
Effect of CSA on secretion of IL-1 by human PBMC. PBMC
were cultured with (B) or without (A) PHA (1 µg/mL) in the presence of the indicated concentrations of CSA or 1 mg/mL of the other GAGs.
After 24 hours supernatants were obtained and IL-1 levels were
determined by ELISA.
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In contrast, the other GAGs (CSB, CSC, and heparin) did not show
comparable effects upon IL-1 secretion. Low-level IL-1 secretion
(over control) was detected when non-PHA-treated PBMC were exposed to
CSB or CSC (1 mg/mL); this cytokine level corresponded to that seen
when cells were treated with a 10-fold lower concentration (0.1 mg/mL)
of CSA (Fig 1A). This low stimulation may be due to the presence of
contaminant CSA in the CSB and CSC preparations (about 10% to 15% in
the form provided by the supplier). An even lower level of IL-1
stimulation was observed for heparin.
Northern blot analysis was performed to determine the effect of CSA on
steady-state IL-1 mRNA levels in PHA-stimulated and unstimulated
PBMC (Fig 2). A substantial induction of
IL-1 mRNA was observed when cells were treated with 1 mg/mL CSA.
This parallels the increase in IL-1 mRNA levels previously
associated with HA treatment in murine macrophages.18
Moreover, an additive effect was observed when CSA and PHA were
combined (Fig 2).
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| Fig 2.
Induction of IL-1 mRNA in human PBMC exposed to
CSA. (A) Total RNA isolated from PBMC cultured in the presence of CSA
(1 mg/mL), PHA (1 µg/mL), or both, was subjected to Northern blot hybridization using IL-1 cDNA as a probe. The positions of the 28S
and 18S ribosomal RNAs are indicated. Relative amounts of RNA loaded in
each lane are visualized in the lower panel by methylene blue staining.
(B) The intensities of the IL-1 hybridization signals shown in (A)
were measured using an InstantImager analyzer, and the results were
plotted as a histogram to highlight the relative IL-1 mRNA levels.
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Because IL-1 is secreted predominantly by monocytes,19
we asked whether the CSA effect could be reproduced in an established monocytic cell line. U937 is a histiocytic lymphoma cell
line20 that can be induced by PMA to differentiate along
the monocytic lineage. In their differentiated state, these cells
secrete IL-1.21,22 CSA induced an approximately 50%
increase in IL-1 release by PMA-treated U937 cells (from 115 pg/mL
to 191 pg/mL in one representative experiment), but had no observable
effect on undifferentiated cells which showed no IL-1 production
(data not shown). This is consistent with the notion that the CSA
effect is dependent on commitment to the monocytic lineage. Monocytes
purified from PBMC were also induced to secrete IL-1 by CSA (from 0 to 115 pg/mL in one representative experiment) (data not shown).
To verify that the observed cytokine effects were indeed attributable
to CSA (and not to impurities, eg, HA, in the CSA preparation), we
digested the commercial CSA preparation before its use with chondroitinase AC, a polysaccharide lyase that acts endolytically on
CSA and CSC. After chondroitinase AC pretreatment, there was only
negligible IL-1 release (Fig 3). When
HA (at 5 µg, a concentration yielding IL-1 secretion equivalent to
1 mg of CSA) was similarly pretreated with chondroitinase AC, its
capacity to induce IL-1 secretion was not affected (Fig 3). Neither
chondroitin sulfate nor hyaluronic acid disaccharide had any effect on
IL-1 secretion (data not shown), suggesting that the observed
activity of the two GAGs is specific for their polymeric forms. These
results taken together indicate that CSA induces monocytes to secrete IL-1, and that this effect is not attributable to impurities.
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| Fig 3.
Effect of chondroitinase AC digestion on the
IL-1-inducing activity of CSA. CSA (10 mg/mL) or HA (50 µg/mL)
was incubated with chondroitinase AC (100 mU/mL) in RPMI (without fetal
calf serum) at 37°C for 2 hours. The reaction mixtures were then
postincubated at 65°C for 10 minutes to inactivate the enzyme. PBMC
were cultured in the presence of untreated and chondroitinase
AC-treated CSA (1 mg/mL) or HA (5 µg/mL) for 24 hours and the level
of IL-1 in the medium was determined as in Fig 1.
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CSB stimulates B-cell proliferation.
Having shown that CSA and HA induce monokine expression, we next looked
for other effects that GAGs might have on PBMC. Cellular aggregation
and proliferation are two commonly studied parameters. Although CSA had
induced IL-1 secretion in our earlier experiments, this GAG had no
observable effects on PBMC proliferation or aggregation (data not
shown). The same held for CSC, heparin, and HA (data not shown).
However, CSB, which had exhibited minimal cytokine effects, induced
both PBMC aggregation (Fig 4) and
proliferation in a dose-dependent manner
(Fig 5A). Aggregation was visible as early
as 2 hours after addition of CSB to cell cultures and increased up to
24 hours. CSB-induced proliferation peaked at day 6 and decreased
thereafter (data not shown). TGF, an anti-inflammatory cytokine
known to have diverse immunoinhibitory activities, completely blocked
this CSB-induced proliferation within the PBMC pool (Fig 5B).
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| Fig 4.
CSB enhances cell aggregation in PBMC cultures. The
effect of (A) control, (B) CSB (1 mg/mL), (C) PHA (1 µg/mL), and (D)
HA (10 µg/mL) on PBMC aggregation is shown. PBMC were cultured in 24-well plates for 24 hours, and the cultures were then examined by
phase microscopy using a Nikon Phase Contrast ELWD 0.3 microscope (original magnification × 100).
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| Fig 5.
Characterization of CSB-induced PBMC proliferation. (A)
PBMC were cultured as described in Materials and Methods, and CSB was
added at the indicated concentrations. (B) PBMC were treated with CSB
(1 mg/mL) in the absence or presence of rTGF1 (1 ng/mL). (C) Total
PBMC or monocyte-depleted PBMC (nonadherent) were incubated with CSB (1 mg/mL) for 6 days. Monocytes were depleted by adherence to tissue
culture flasks and nonadherent cells were collected, counted, and
plated. Data shown are [3H]-thymidine incorporation
during the final 16 hours of a 6-day culture period. In each case, the
results shown are representative of at least three similar
experiments.
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To determine the effect of CSB on the proliferation of individual cell
types within the PBMC population, monocytes were depleted by adherence
to tissue culture flasks, and the residual nonadherent cells were
stimulated with CSB. Although this nonadherent cell fraction aggregates
when stimulated with CSB, the cells do not proliferate (Fig 5C).
Proliferation could be restored when monocytes were reintroduced into
the cultures (data not shown). No significant [3H]-thymidine incorporation was observed when monocytes
alone were stimulated with CSB (data not shown). In addition, purified
T cells did not proliferate when stimulated with CSB alone or in combination with PMA or CD3 antibody (OKT3), or in the presence of
monocytes (data not shown). These results suggest that CSB-induced proliferation is monocyte dependent.
To further dissect the effect of CSB on PBMC, we attempted to determine
the particular cell population responding to CSB. PBMC were stimulated
with either CSB, HA, or PHA. Six days later, cultures were analyzed for
expansion of CD4+, CD8+, and   T cells and
CD19+ B cells by flow cytometry. Although CSB did not
stimulate the different T-cell subsets, it did induce blast
transformation (Fig 6A) and expansion (Fig
6B) of CD19+ B cells. This effect was specific to CSB, as
it was not observed with HA or PHA. The expansion and blast
transformation of CD19+ cells as a response to CSB was
blocked in the presence of TGF1 (Fig 6A and B), consistent with the
TGF1 inhibitory effect on CSB-induced PBMC proliferation (Fig 5B).
Of note, TGF1 inhibited in a dose-dependent fashion both CSA- and
HA-induced IL-1 secretion (data not shown). These results establish
that CSB is a B-cell activator and that its activity is monocyte
dependent and TGF1 inhibitable.
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| Fig 6.
Blast transformation and outgrowth of human peripheral
blood B cells after stimulation with CSB. Control PBMC (1 × 106/mL) or PBMC stimulated with either CSB, HA, PHA, or CSB
in combination with rTGF1 (1 ng/mL) were cultured for 6 days and
then immunostained with phycoerythrin-conjugated CD19 MoAb and analyzed
on a FACScan. Two-dimensional plots were generated in which
CD19-positivity was plotted on the x-axis and side scatter (as a
reflection of cell size) was plotted on the y-axis. In (A), the
percentage of CD19+ cells showing blast transformation
(as reflected in a size exceeding a threshold value established by
control unstimulated cells) has been calculated. In (B), the overall
percentage of CD19+ cells detected in these plots is
shown. Nonspecific binding was controlled by using appropriate
isotype-matched antibodies and was the same for all cultures.
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CSB proliferative effect is CD44 dependent.
HA's immunomodulatory effects on murine macrophages (IL-1
induction)18 and B cells (proliferation and
differentiation)23 have been shown to be CD44 dependent.
Although CD44 principally binds to HA, it appears to also interact at a
more modest level with CS.24 The hematopoietic CSPG,
serglycin, was identified as a ligand for CD4425 and is
involved in lymphoid cell activation.8 Furthermore, the CS
form of the invariant chain (associating with MHC class II) can enhance
T-cell responses through the interaction with CD44.7 To
determine whether CD44 is also involved in CSB induction of cell
proliferation, PBMC were exposed to CSB in the presence of 10 µg/mL
of CD44 MoAb (A3D8). Addition of the CD44 antibody A3D8 significantly
blocked CSB-induced [3H]-thymidine incorporation
(Fig 7A). However, this antibody did not
inhibit or stimulate [3H]-thymidine incorporation when
added alone or with any concentration of PHA26 (and data
not shown), ruling out toxic effects. Moreover, a CD44-Rg fusion
protein24 inhibited CSB-induced PBMC proliferation (Fig 7B). Although HA is the principal known CD44 ligand, it did not induce
[3H]-thymidine incorporation by itself and did not
diminish CSB-induced proliferation when added simultaneously in
proliferation assays (data not shown). Therefore, CSB seems to induce
B-cell proliferation via CD44, independent of HA ligation.
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| Fig 7.
CD44 MoAbs and CD44-Rg inhibit CSB-induced PBMC
proliferation. PBMC were incubated with CSB (1 mg/mL) with or without
either the CD44 MoAb A3D8 (10 µg/mL; A), or CD44-Rg fusion protein
(12 mg/mL ; B) for 6 days. [3H]-thymidine incorporation
was measured in the last 16 hours of the experiment. The data were
confirmed in three independent experiments.
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These results suggest novel functions for CSA and CSB as accessory
molecules during monocyte and B-cell activation, respectively. Moreover, the data are consistent with CSB functioning through a
cell-specific CD44 pathway that is not accessed by HA.
| |
DISCUSSION |
Soluble PGs arise at inflammatory sites by secretion from activated
monocytes/macrophages and via the degradation of extracellular matrix.5 In turn, PGs, whether soluble or cell-associated, have been functionally linked in a number of isolated reports to immune
cell activation and inflammatory processes. The chondroitin 4-sulfate
type of serglycin, a CSPG secreted by hematopoietic cells,5
is a ligand for CD44 and contributes to the activation of cytotoxic
T-cell clones.8 In another study bearing upon T cells, the
chondroitin sulfate form of invariant chain (associated with MHC class
II) was shown to enhance the stimulation of T cells via interaction
with CD44.7 A connection has also been established between
PGs/GAGs and macrophage activation. Soluble HA induces the expression
of IL-1, tumor necrosis factor- , and IGF-1 in macrophages,17,18 and this expression can be blocked by
CD44 MoAbs.18
The present study establishes additional links between the CSs and
selected immune cell populations. Significant findings include: (1)
CSA, like HA, stimulates monokine secretion by PBMC; (2) CSB, another
chondroitin 4-sulfate, stimulates aggregation of PBMC and promotes
monocyte-dependent B-cell blast transformation and proliferation,
which is inhibitable by both the anti-inflammatory cytokine TGF and
CD44 MoAbs; and (3) CSA and CSB do not appear to overlap in their
immunomodulatory activities, and neither CSC, a chondroitin 6-sulfate,
nor heparin share in these activities. The data suggest yet another
extrinsic pathway leading to the triggering of inflammatory monokine
production. Moreover, the data constitute the first direct connection
between CSs, and in particular chondroitin 4-sulfates, and both the
monocytic and B-cell compartments. Hence, the CSs, the main GAGs
secreted by activated monocytes, emerge here as a potentially
significant class of inflammatory mediators.
CD44 has previously been implicated in mediating HA-induced activation
of human and murine monocytes,17,18 as well as in a recent
report, murine B cells.23 Moreover, CD44 has also been implicated in CS-induced activation of T cells.7,25 The
findings of the present study significantly expand on these previous
CD44-related observations. Our data offer the first evidence that CD44
is involved in human B-cell activation. Furthermore, the present
findings show that CD44 is functionally required for CS-induced, and
not just HA-induced, activation of monocytes and B cells. While the present data indicate that CD44 is somehow involved in B-cell activation, they do not formally distinguish between the possibilities that the functional CD44 is on B cells versus interacting monocytes, a
question prompted by our additional observation that the CSB effect on
B-cell proliferation is monocyte dependent. However, it is noteworthy
in this regard that previous reports have clearly documented increased
CD44 expression after B-cell activation, with associated changes in the
CD44 isoforms expressed.27-29 Moreover, HA-induction
appears to operate directly on the CD44 of B cells in the murine
system.23 Additionally, our data showed no effect of CSB on
human monocyte activation (monokine production). Taken together, it
seems most likely that the CSB effect on B cells observed here in the
human system is mediated by the B cell's CD44.
Although studies have tended to emphasize HA as a principal ligand for
CD44, there is in fact evidence that CS also binds to CD44, albeit at a
100-fold lower concentration than does HA.24 In line with
this previous binding data, our functional data showing a
CD44-dependent CSB-induced effect on B-cell proliferation is likely
explained by direct CSB binding to CD44 (on B cells and/or monocytes). However, the data do not rule out other mechanisms. It is
also tempting to speculate that the CSA effect on monokine secretion,
like the CSB effect on B-cell activation, may be CD44 dependent. This
would fit with our observation that CSA stimulates IL-1 secretion by
PBMC at a 100-fold excess of CSA as compared with HA, mirroring the
differences between CS and HA in their CD44-binding capacities.
However, we could not address this interesting question because the
CD44 MoAb used here demonstrates agonist activity that induces cytokine
secretion by monocytes (data not shown).
The functional differences observed for the various GAGs examined in
this study are of special interest. CSA and CSB, both chondroitin
4-sulfates, exhibited markedly different functional profiles, with
their primary effects directed toward monocytes and B cells,
respectively. Even more strikingly, HA, notwithstanding its high
reported binding affinity for CD44, did not share CSB's B-cell
modulatory activities nor did it compete with CSB. These differential
effects observed here for CSA and HA on monocytes and CSB on B cells
might stem from the expression of distinct CD44 isoforms and
conformational types with different GAG-binding capacities on these
cells.24,27,30-36 Further study of the ability of the
variant forms of CD44 expressed during B-cell development and their
relative ability to bind CS is clearly warranted.
An important aspect of the present study is the use of whole PBMC pools
rather than purified cell subpopulations or cell lines. This
experimental approach permits the detection of immunological effects
that are dependent on cell:cell interactions, and in our case, was
essential for detecting the CSB-dependent B-cell proliferation in the
first place because it appears to be dependent on the presence of
monocytes. Just how monocytes participate in this phenomenon, as well
as how CSB and CD44 play into this, remains to be determined.
B cells can be activated in T-independent fashion using polymeric
molecules that bear repeating epitopes. CSB, being composed of
repeating disaccharide units, is in principle equipped to provide the
first signal for such cross-linkage-dependent B-cell activation. The
second signal could come from monocytes, explaining the observed monocyte dependence in our system. Furthermore, the inherently higher
valency associated with PGs, as compared with free GAGs, might confer
upon them higher functional efficacy, both for CSA-containing ones (in
the case of monocyte triggering) and for CSB-containing ones (in the
case of B-cell triggering). Hence, although relatively higher
concentrations of free CS GAGs (as compared with the HA GAG) are needed
to achieve functional immunostimulation in the experimental assays used
here, they likely underestimate the potency of these GAG stimulators
when PG based.
The present study suggests at least two ways in which GAGs could
contribute to inflammatory processes. First, GAGs could tie into
pro-inflammatory cytokine secretion. This appears to be the case for
both CSA and HA, which can trigger the secretion of monokines. The
induction of IL-1 documented here is especially significant in light
of the established pivotal role of this cytokine in the inflammatory
cascade.19 Second, GAGs could influence lymphoid cells
directly, without cytokines as intermediaries. Our studies highlight
this point for the CS:B-cell interface. Unravelling of a GAG
"pro-inflammatory pathway" could provide novel therapeutic targets within inflammatory sites in pathophysiologic settings. For
example, in the case of CSA, there is the potential for therapeutics that intercept this local molecular trigger of IL-1 secretion, as an
alternative to other therapies under development (eg, soluble IL-1
receptors and IL-1 receptor anatagonists) which deal with blocking
IL-1's downstream effects. More information pertaining to soluble
PG and free GAG accumulation in pathophysiologic settings will first be
needed.
| |
FOOTNOTES |
Submitted October 20, 1997;
accepted March 5, 1998.
Supported in part by grants from the National Institutes of Health (NIH
RO1 AI38960, NIH RO1 CA74958, and NIH RO1 AI31044).
Address reprint requests to Mark L. Tykocinski, MD,
Department of Pathology, Biomedical Research Building Room 925, Case
Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr A. Hochberg, Dr D. Cibrik, and G. Riely for helpful
discussions and review of the manuscript. We appreciate Dr M. Lamm's
support throughout this project. We also thank Susi Brill for her
expert secretarial assistance.
| |
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Abstract
Introduction
Abstract