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Departments of
*
Medicine,
Pathology, and
Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital, and
Division of Infectious Disease, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
(8, 10, 11, 12, 13, 14, 15). Polysaccharides have been believed to be
relatively thymus-independent Ags (TI-2 Ags) that activate B cells by
extensively cross-linking their membrane-bound Ig receptor to induce Ab
production. Although six to seven sugar residues have been identified
as sufficient to fill the Ag-binding site of Igs
(16, 17, 18), the affinity of Ab binding of some
polysaccharides has been positively correlated with increased chain
length, which allows full expression of putative conformational
epitopes (19, 20, 21, 22, 23, 24). Besides antigenicity, in humans the
immunogenicity of polysaccharides is known to be a chain
length-dependent phenomenon. Polysaccharides with a molecular mass
below 80,000–90,000 Da do not elicit an Ab response
(25).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. fragilis NCTC 9343 was originally obtained from the National Collection of Type Cultures (London, U.K.). After rat-spleen passage, the bacteria were stored at -80°C in peptone-yeast broth until use and then grown anaerobically as previously described (27, 28). PS A was extracted from the bacterial cells by hot phenol/water; digested extensively with RNase, DNase, and pronase/protease K; and purified by gel filtration in a 3% deoxycholate acid-containing buffer at pH 9.8. Further purification was achieved by gel filtration and anion-exchange chromatography as previously described (27, 28). The polysaccharide was subjected subsequently to isoelectric focusing to disaggregate this highly charged molecule. The final product was found to be essentially free of contaminating protein, nucleic acid, LPS, and lipids by the following assays: UV-scanning, protein measurements including the Lowry (29) and BCA (Pierce, Rockford, IL) methods, silver stain SDS-PAGE gels (30), and high resolution (500 MHz) proton nuclear magnetic resonance (NMR) spectroscopy (9, 10, 11, 27, 28).
Ozonolysis and purification of depolymerized PS A
PS A was suspended at a concentration of 5.93 mg/ml in buffer consisting of 900 µl of 0.01 M PBS with 0.05% NaN3 (pH 7.2) and 100 µl of 0.2 M NaHCO3 (final pH 7.6) (PBSA). Ozone was generated from compressed air through an ozone generator (More-Zon10, Tapei, Taiwan). A stream of ozone/air gas (21% O3) was passed through the solution at room temperature at a flow rate of 3.17 ml/s. At time interval 0 and at various subsequent time intervals, 35 µl of the reaction solution was removed and diluted with 35 µl of PBSA, and the molecular size distribution of the products was determined by gel filtration chromatography on Superose 6, Superose 12, and Superdex 30 columns calibrated with dextran standards according to the fractionation ranges of the gel (Amersham Pharmacia Biotech, Piscataway, NJ) at a flow rate of 0.5 ml/min (26, 31). The columns were eluted with PBSA, and the fractions were monitored by a differential refractometer. The ozone flow was terminated when products reached the desired molecular size. An aliquot of the depolymerized PS A was removed, and the reaction was then allowed to continue. The depolymerized saccharide was separated from native PS A by gel filtration chromatography on a Superdex 200 column (Amersham) equilibrated in 0.3 µM sodium phosphate buffer (pH 7.2) with 0.05% sodium azide. The fractions were again monitored by a differential refractometer. The polysaccharide of the desired size was desalted by gel filtration with G25 media (Amersham), lyophilized, and stored in 3 M NaCl at a concentration of 5 mg/ml at -20°C to avoid reaggregation.
NMR
Proton NMR experiments with native and depolymerized PS A were performed on a Bruker AMX500 spectrometer with a proton resonance frequency of 500.13 MHz. All 1H spectra were recorded at 70°C in 2H2O, and chemical shifts were referenced in relation to H2HO resonance at 4.26 ppm.
T cell proliferation assays
Mononuclear cells and
CD4+CD8- cells from
Leukopacks from different anonymous platelet donors were isolated by
centrifugation of cells in Ficoll-Hypaque gradients and purified using
nylon wool and immunomagnetic beads as previously described
(32, 33, 34). The purity of the
CD4+CD8- cell population
was confirmed by FACS analysis (
95%). Gamma-irradiated mononuclear
cells (1 x 105/well), which served as APCs,
were incubated in quadruplicate at 37°C, 5%
CO2 with
CD4+CD8- lymphocytes
(5 x 104/well) plus PS A of 129.0, 77.8,
46.9, 17.1, 5.0 kDa (20 µg/ml) or with medium only (as a negative
control) in a 96-well plate with RPMI 1640 complemented with
L-glutamine, sodium pyruvate, penicillin-streptomycin,
nonessential amino acids, 2-ME (Life Technologies, Gaithersburg, MD),
and 10% FBS (Intergen, Purchase, NY). The quantity of APCs and
CD4+ cells, as well as the Ag concentration, were
shown to be optimal in preliminary experiments. Earlier studies had
revealed maximal T cell proliferation at day 8 with continuous
stimulation by native PS A. Therefore, after 8 days, cells were pulsed
with [3H]thymidine (1 µCi/well) for 6 h
and harvested with a semiautomated cell harvester. Incorporation of
[3H]thymidine by
CD4+CD8- T cells was
quantitated in a scintillation counter, and the results were expressed
as cpm. Three independent T cell proliferation assays, each with
CD4+ cells from different donors, were performed
for PS A of each molecular size.
Animal model of intraabdominal abscess formation
An animal model of intraabdominal abscess formation was used to evaluate the effect of molecular size on protection against abscess formation (35). In two independent experiments, groups of 10 outbred male Wistar rats (150–175 g; Charles River Laboratories, Wilmington, MA) were injected s.c. with a sterile solution of 100 µl of 0.15 M PBS containing 10, 1, or 0.1 µg of PS A of 129.0, 46.9, 17.1, or 5.0 kDa; 100 µl of 0.15 M PBS were used as a negative control. Each group was treated with polysaccharide at -24 h, 0 h (challenge), and +24 h. The challenge inoculum contained B. fragilis NCTC 9343, sterile cecal contents, and 10% barium sulfate. Another group of five animals was challenged with sterile cecal contents and 10% barium sulfate alone. Rats were anesthetized with a single i.p. injection of 0.15 ml of Nembutal (50 mg/ml; Abbott Laboratories, North Chicago, IL). An anterior midline incision (0.5 cm) was made through the abdominal wall and peritoneum, and a gelatin capsule with 0.5 ml of inoculum was inserted into the pelvis (36). Six days after challenge, the rats were sacrificed and examined for macroscopically visible i.p. abscesses by observers who were unaware of treatment status. Grossly visible abscesses were confirmed microscopically. The development of one or more abscesses was considered a positive result.
Statistical analysis
For PS A of each molecular size and negative controls, three independent T cell assays were performed, each in quadruplicate. The stimulation ratio for a saccharide was defined as the ratio of the cpm of the stimulated T cells in one well divided by the geometric mean of the cpm for the corresponding four wells with the negative controls. For each chain length, each assay yielded four correlated stimulation ratios. Log stimulation ratios were analyzed by clustered-measures analysis of variance (37).
In vivo experiments were analyzed in a structured logistic regression
model that permitted evaluation of separate dose-response relationships
(dose in micrograms and moles) and direct interference on median
abscess protective dose. Specifically, Pr (abscess/dose = 
,
length = 
) = 
{1 + exp[
· ( -
)]}-1, where
> 0 is the rate of abscess formation in positive controls,
is the slope of the dose-response relation
on the logistic scale for chain length , and
is the median protective dose
(PD50 = dose that would have protected 50% of
the saline-treated rats from abscess formation) for the chain length
(38). This analysis accommodated the fact that not all
positive control animals developed abscesses. Likelihood ratio tests
were performed for hypotheses concerning commonality of dose-response
slopes and PD50s. Rats that died within 2 days after challenge were not
included in the analysis because their deaths were due to anesthesia
and surgery.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Native PS A with an average molecular size of 129.0 kDa (upper
limit, 245.5 kDa; lower limit, 74.3 kDa), which corresponds to
approximately 155 repeating units, was oxidized with ozone to produce
PS A polymers of decreased molecular size. After treatment for 6, 8,
11, 16, 21, and 23 min, the resulting average polysaccharide sizes were
determined by gel filtration chromatography (Fig. 2). The kinetics of the decrease in
polysaccharide size following ozone treatment appeared to fit a
logarithmic function. The molecular size distribution of the ozonolysis
products were typical of polysaccharide preparations, eluting in one
symmetrical peak (data not shown). Samples corresponding to the
generation of PS A with the average molecular sizes of 129.0, 77.8
(upper limit, 204.2 kDa; lower limit, 20.5 kDa), 46.9 (upper limit,
155.0 kDa; lower limit, 11.8 kDa), and 17.1 kDa (upper limit, 77.2 kDa;
lower limit, 4.2 kDa) were used for the biological assays. Efforts to
generate PS A of 
10 kDa yielded (at 54 min) an oligosaccharide
with an average molecular size of 5 kDa (upper limit, 21.5 kDa; lower
limit, 1.6 kDa) that corresponded to 6 repeating units. Proton NMR
analysis of native and depolymerized PS A chain lengths showed the
saccharides to have intact repeating units (Fig. 3).
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PS A with average polymer sizes of 129.0, 77.8, 46.9, and 17.1 kDa
elicited T cell proliferation significantly greater than that in the
negative control. The stimulation ratios of the quadruplicate
experiments in three independent assays showed that PS A as small as
17.1 kDa was as potent in inducing T cell proliferation as was PS A of
larger molecular size (p = 0.76) (Fig. 4A). Because of this result, a
smaller chain-length size of PS A was generated and tested in T cell
proliferation assays. To accomplish this, native PS A from the same lot
was prepared by isoelectric focusing and then depolymerized by
ozonolysis into 5.0-kDa average molecular sized molecules. The native
PS A (Fig. 4B) was shown to have the identical structure and
purity as the native PS A produced previously (Fig. 3
) from this lot.
The 5.0-kDa polysaccharide was structurally intact (Fig. 3) but was
significantly less stimulatory to CD4+ T cells
than native PS A (p = 0.035) (Fig. 4B)
(9)4. The
ability to induce T cell proliferation was dose dependent (data not
shown).
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PS A of 129.0 (from Fig. 4A), 46.9, 17.1, and 5.0 kDa
was tested for the ability to protect rats against B.
fragilis-induced abscess formation. In these experiments, 87.5%
of saline-treated rats developed intraabdominal abscesses (Fig. 5). None of the control rats (challenged
with barium sulfate and sterile cecal contents only) developed
abscesses (data not shown). After B. fragilis challenge,
21%, 26.3%, and 31.6% of rats treated with 10 µg of 129.0-kDa PS
A, 46.9-kDa, and 17.1-kDa PS A, respectively, developed abscesses. Of
rats treated with 10 µg of 5.0-kDa PS A, 68% developed abscesses.
Protection was dose dependent.
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shows that the required dose of 5.0-kDa
PS A is well beyond the upper limit of doses used in this
experiment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three biologically critical structural features of PS A needed to be
preserved on depolymerization: 1) the galactofuranose side chain; 2)
the acid-labile 4,6-pyruvate structure; and 3) the very reactive free
amino group (39). Traditional methods would have destroyed
these biologically active groups. Enzymes specific for the glycosidic
linkages in the backbone of PS A have not yet been described.
Therefore, ozonolysis, which was shown to depolymerize polysaccharides
by cleaving specifically -D-aldosidic linkages,
represented a promising alternative. In preliminary studies in which we
used the previously described method, including peracetylation and
deacetylation, to achieve the most selective oxidation, we obtained
smaller structurally intact polymers but found it impossible to
selectively deacetylate the molecule to regain the positive charge in
only a single position. Without the protection of possibly reacting
groups, two -D-aldosidic linkages were at risk of
oxidization: one between the galactofuranose side chain and the
galactopyranose of the backbone, and the other between the
galactopyranose bearing the pyruvate group and the fucose of the
backbone. As confirmed by NMR spectroscopy, the
-D-aldosidic linkage of the galactofuranose side chain
was less reactive to ozonolysis, a result suggesting that
ozonolysis, even without protection of the hydroxyl groups,
specifically cleaved -D-aldosidic linkages of backbone
sugars under stereoelectronic control (Fig. 2) (26).
In this study, characterization of the T cell response to PS A of different chain lengths demonstrated that the biological activity of the saccharide resides in a polymer as small as 17.1 kDa. Our statistical analysis took into account the fact that the three independent T cell assays for PS A of each molecular size were performed with CD4+ cells from different donors. Despite the individual variability of the response, PS A molecules with average molecular sizes between 129.0 and 17.1 kDa stimulated CD4+ cells to a similar degree (p = 0.76). The 5.0-kDa PS A was significantly less capable of stimulating T cell proliferation than its native polymer (p = 0.035). These in vitro results correlated with results in vivo: the 129.0-, 46.9-, and 17.1-kDa PS A molecules were equally able to protect rats against abscess formation (PD50 = 0.73 µg), whereas 5.0-kDa PS A was not protective (PD50 = 187.1 µg, p < 0.001).
In the context of what is known about the influence of the size of T
cell-independent polysaccharides on immunogenicity, our results
strengthen the conclusion of earlier in vivo experiments classifying PS
A as a T cell-dependent Ag and clearly distinguish this molecule from
polysaccharides with only negatively charged or no charged residues.
Like that of other T cell-independent polysaccharides, the
immunological activity of PS A correlates with its molecular size.
However, in contrast to these other polysaccharides, PS A retains its
full antigenicity at least down to a size as small as 17.1 kDa. T
cell-independent saccharides investigated, in particular, dextrans and
capsular polysaccharides from group B streptococci, Streptococcus
pneumoniae, Neisseria meningitidis, and
Haemophilus influenzae, are thought to bind membrane-bound
Ig receptor on B cells to induce a humoral response. Affinity of Ab
binding was shown to be positively correlated with increasing chain
length. Up to hundreds of repeating units in the case of group B
streptococcal and S. pneumoniae type 14 capsular
polysaccharides are necessary to elicit optimal binding activity, thus
supporting the hypothesis that anticapsular Abs recognize a
conformational epitope fully expressed only in high m.w. forms of the T
cell-independent saccharide molecules (21, 24, 40).
Although six to seven sugar residues of a linear 
(1
6)-linked
glucose have been proven sufficient to fill the Ag-binding site of Igs
(16, 17, 18), such small-sized saccharides do not induce
humoral response in primates. Nonzwitterionic polysaccharides smaller
than 80,000– 90,000 Da do not induce Ab response (25),
and even T cell-independent polysaccharides with a molecular size
between 80,000 Da and 200,000 Da are only variably immunogenic in
humans (40). Our findings with PS A clearly distinguish
this molecule from those T cell-independent polysaccharides.
The mechanism through which T cells are stimulated by a zwitterionic
polysaccharide is not known, but the results suggest two possible
pathways. Since APCs and a minimal PS A size of 7–22 repeating units
are necessary to stimulate CD4+ cell
proliferation, we hypothesize that the Ag-presenting molecules on APCs
and the signaling molecules on CD4+ cells are
linked by zwitterionic polysaccharides. In the first of the two
possible scenarios, if PS A were presented (for example) in the binding
groove of a MHC class II molecule (with or without being processed), it
would need to have, depending on its primary, secondary, tertiary, and
quaternary organization, a minimal molecular size between 1602 Da and
about 2996 Da to fit into the cleft (41, 42, 43, 44, 45, 46, 47, 48). Conceivably,
hydrogen bonds between charged atoms along the PS A and residues of the
-helical regions and between PS A and the -sheet of HLA-DR (for
instance) are a component of the binding interaction that is essential
to T cell activation. The conformation of PS A might allow most of its
charged residues to get into contact with the T cell receptor. The
ability of PS A to interact with T cells from different donors
(different haplotypes) suggests promiscuous binding motif(s) due to the
existence of different epitopes in the PS A molecules that bind
different MHC determinants. In the second scenario, a zwitterionic
polysaccharide might act as a T cell mitogen. For instance, for PS A to
interact with both MHC and T cell receptor molecules outside their
normal Ag-specific sites (in a manner similar to that by which
superantigens induce T cell activation), a minimal size would be
required. Most of the known superantigens have a molecular mass between
15,000 and 40,000 Da (49, 50, 51, 52, 53, 54, 55, 56). Superantigens bind as
intact proteins, and no large conformational change occurs upon complex
formation (50). However, in comparison to the binding of
peptides in the MHC class II cleft, hydrogen bonds seem to play a minor
role in the binding of a superantigen to its ligand.
These results demonstrate that a small zwitterionic polysaccharide, successfully generated by ozonolysis, comprising 22 repeating units fully elicits cell-mediated immunological response. These findings clearly distinguish this molecule from T cell-independent polysaccharides and give evidence of the existence of a novel mechanism for a polysaccharide-induced immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dennis L. Kasper, Channing Laboratory, 181 Longwood Avenue, Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: PS, polysaccharide; NMR, nuclear magnetic resonance; PD50, dose that would have protected 50% of the saline-treated rats from abscess formation; CI, confidence interval.
4 Although the native PS A used in experiments shown in Fig. 4, A and B, is from the same lot, the quantitative level of stimulatory activity of the control native polysaccharide differed in the two sets of experiments. Isoelectric focusing is used to disaggregate the polysaccharides. The results shown in Fig. 4B were obtained with PS A used shortly after isoelectric focusing. We believe the T cell-stimulatory activity of this material was greater than that seen in Fig. 4A because testing quickly followed this disaggregation step and the polysaccharides were used promptly in T cell proliferation assays. We have found that these zwitterionic polysaccharides aggregate very quickly in storage, resulting in reduced T cell-proliferative activity (data not shown). Because of the length of the time required for preparation of different chain length sizes of PS A used in Fig. 4A, these polymers had less in vitro activity.
Received for publication August 5, 1999. Accepted for publication November 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-D-aldosidic linkages. Proc. Natl. Acad. Sci. USA 95:6584.
+ T cells respond to mycobacterial heat-shock protein. Nature 340:309.[Medline]
/ T cells. J. Exp. Med. 186:899.-chain does not require processing. J. Immunol. 140:1063.[Abstract]
chain of known three-dimensional structure. J. Exp. Med. 182:1833.-chain complexed with a superantigen. Nature 384:188.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 1 µg (•), and 0.1 µg (
)
of PS A of the molecular sizes noted on the x-axis;
control rats were treated with saline (
). Vertical displacements of
symbols indicate proportion of rats developing intraabdominal
abscesses. The lines depict structured logistic regression fit assuming
a common logistic slope for all lengths. PS A of 17.1, 46.9, and 129.0
kDa (average molecular sizes) shared a PD50 value (0.73
µg), while 5.0-kDa PS A (average molecular size) had a distinct and
extremely high PD50 (187.1 µg, p <
0.001).