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*
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115; and
Neose Technologies Inc., Horsham, PA 19044
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ex
vivo, compared to peritoneal cells from mice injected with Dex.
Gr1+ cells adoptively suppressed naive CD4+ T
cell proliferation in vitro in response to anti-CD3/CD28 Ab
stimulation. Suppression of naive CD4+ cells involved cell
contact and was dependent on IFN-
and NO, with a discrete role
played by IL-10. Coculture of naive CD4+T cells with
Gr1+ suppressor cells did not lead to CD4+ T
cell apoptosis, although it did imprint on naive CD4+ T
cells a response characterized by lower levels of IFN-, coincident
with increased IL-13 production. Our results suggest that both human
milk and helminth parasites may share a ligand-specific mechanism
involved in the generation of anti-inflammatory mediators that suppress
Th1-type and inflammatory responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by
innate immune cells responding to pathogen-expressed molecules termed
"pathogen-associated molecular patterns" (1, 2, 3, 4, 5). In
contrast, helminth infection generally biases the immune response
towards a Th2-type profile and also may be associated with induction of
Ag-specific or nonspecific anergy (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). To date, other
than studies on implantation of filaria in mice that demonstrate an
IL-4-dependent, cell contact mechanism between regulatory and responder
T cells, there is scant other information regarding how helminths drive
Th2 biasing or induce suppression other than the nature of
immune-biasing Ags (16). In this regard,
helminth-expressed molecules that drive Th2-biasing and/or
immunosuppressive responses generally have been reported as
excreted/secreted products from Nippostrongylus,
Toxocara, Schistosoma, and Taenia
(7, 17, 18, 19).
Among helminths, schistosomes are potent Th2 drivers and inducers of
immune anergy, with the parasite egg as the strongest stimulus of these
responses. Of the myriad components present in schistosome eggs,
carbohydrates have been shown to be largely responsible for inducing
Th2, anti-inflammatory, and IgE responses (19, 20).
Further, we identified two schistosome oligosaccharides,
lacto-N-fucopentaose III
(LNFPIII)2 and
lacto-N-neotetraose (LNnT), that are Th2-biasing and capable
of suppressing Th1 responses in vivo (21, 22). These two
oligosaccharides also are found in human milk, which has been shown to
be immunosuppressive (23). In this study, we asked if the
immunomodulatory properties of the glycoconjugate LNnT-Dex, are
attributable in part to activation of innate mechanisms that contribute
to Th2 biasing and immune suppression. We found that injection of mice
with LNnT-Dex led to a rapid "innate" expansion of cells expressing
the surface markers Gr1, CD11b, and F4/80. This Gr1+
population suppressed the proliferative response of naive
CD4+ T cells to primary stimulation with anti-CD3 and
anti-CD28 Abs, by a mechanism that required both cell-to-cell
contact and soluble factors. In addition, Gr1+ suppressor
cells produced low levels of proinflammatory cytokines coincident with
elevated levels of IL-10 and TGF-. Lastly, and perhaps most relevant
to a mechanism that leads to a possible Th2 biasing and
antiinflammatory responses, we demonstrate that LNnT-Dex
Gr1+ suppressor cells were able to "imprint" a Th2
phenotype on otherwise naive CD4+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 10-week-old female BALB/c and C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained in a pathogen-free environment at the Harvard Medical School animal facility in accordance with institutional guidelines.
Glycoconjugate injection and collection of PECs
LNnT-Dextran (LNnT-Dex) was conjugated to a 10 kDa molecule of dextran and was supplied by Neose Technologies (Horsham, PA). Three different glycoconjugates were used, having 21, 35, or 45 molecules of LNnT per molecule of dextran.
LNnT-Dex 21, 35, or 45, diluted in saline, was administered at a dose of 100 µg/mouse in all experiments. Naive BALB/c or C57BL/6 mice were injected i.p. with the LNnT-Dex, dextran, or saline, and animals were sacrificed 2 and 18 h later by CO2 inhalation.
Peritoneal exudate cells (PECs) were obtained at 2 and 18 h after injection by peritoneal lavage with 5 ml of ice-cold HBSS (Life Technologies, Rockville, MD). PECs were washed two times, and RBC were lysed by hypotonic shock with ammonium chloride. Viable cells were counted and adjusted to 5 x 105 cells/ml. Viability measured by trypan blue exclusion was routinely over 95%. PECs were analyzed for surface markers, cytokine production, and suppressor activity in cocultures with naive CD4 cells.
Flow cytometric analysis
Peritoneal exudate cells were blocked with anti-mouse FcR
Ab (CD16/CD32) and stained with FITC-conjugated mAbs against Mac-1
(CD11b), F4/80, Gr-1 (Ly-6G), MHC-II, B7-2, or PE-conjugated Abs
against Gr-1 (Ly-6G), and IL-10R. All Abs were purchased from BD
PharMingen (San Diego, CA), except anti-F4/80, which was obtained
from Serotec (Oxford, U.K.). Stained cells were analyzed on a
FACSCalibur with CellQuest software (BD Biosciences, Mountain View,
CA). Live cells were electronically gated with forward and side scatter
parameters.
Cell culture and coculture of PECs-CD4+ cells
All cultures and cocultures were maintained in RPMI 1640 (Life
Technologies) supplemented with 2 mM L-glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin (Sigma, St. Louis, MO), 5 x
10-5 M 2-ME (Life Technologies) and 10% FBS (Hyclone
Laboratories, Logan, VT). PECs were adjusted to a concentration of
5 x 105/ml, plated in 24-well plates (Costar,
Cambridge, MA) and were maintained at 37°C and 5% CO2
for 72 h. Supernatants were harvested, centrifuged, and examined
for spontaneous production of IFN-, IL-12, IL-10 (Abs and
cytokines were obtained from BD PharMingen), IL-1, IL-18, IL-13, and
TGF- (obtained from R&D Systems, Minneapolis, MN).
Coculture of PECs with naive CD4+ cells was performed as
follows: PECs were obtained as described and adjusted to 5 x
105 PECs/ml. Splenocytes were prepared from naive mice and
enriched for CD4+ cells (>95% by FACS analysis) with CD4
MACS (Miltenyi Biotec, Bergisch Gladbach, Germany).
CD4+ cells were plated in 96-well flat-bottom plates
(Costar) that were precoated with anti-CD3 and anti-CD28 Abs
(BD PharMingen) at 1 µg/ml. Three hours later, PECs were added to
CD4+ T cells at ratios of from 1:4 to 1:16
(PECs:CD4+). Cultures were maintained at 37°C and 5%
CO2 for 72 h, then 1 µCi/well
[3H]thymidine (185 GBb/mmol activity, Amersham,
Aylesbury, U.K.) was added and incubated for a further 18 h. Cells
were harvested on a 96-well harvester (Tomtec, Toku, Finland) and then
counted with a beta-plate counter. Values are represented as cpm from
triplicate wells. In some experiments, supernatants from cocultures
were harvested and analyzed for IL-4, IL-13, and IFN-
production.
Removal and enrichment of Gr1+ cells
The Gr1+ cell population of PECs was removed with MACS beads. Briefly, PECs pooled from four mice were incubated with monoclonal anti-Gr-1 Ab (BD PharMingen) for 30 min at 4°C, washed two times, and reincubated with paramagnetic beads (MiniMACS, Miltenyi Biotec) coupled to goat anti-rat IgG (isotype of the anti-Gr-1 Ab) for 15 min at 4°C. PECs were washed twice and passed through a magnetic column (Miltenyi Biotec) to retain Gr1+ cells. The eluted cell fraction (<5% Gr1+ according to FACS analysis) was adjusted to 5 x 105 cells/ml and used in the cocultures as described above. The positive fraction was obtained and passed again through a new magnetic column; cells obtained here were 75% positive for Gr-1 according to FACS analysis. This population was adjusted to 5 x 105 cells/ml and used in the cocultures as described above. Furthermore, both positive and negative fractions were analyzed for morphology by cytospin as described previously (15) and stained with Giemsa stain (Sigma).
Fixed PECs, transwells cultures, and blocking Ab experiments
In some cocultures, PECs were fixed with 0.5% paraformaldehyde for 5–10 min, washed extensively with RPMI 1640, adjusted to 5 x 105/ml, and added in varying concentrations to 96-well plates in the presence of CD4+ naive cells previously stimulated with plate-bound anti-CD3/CD28 Abs. These cocultures were processed as described previously.
In other cultures, 24-well (0.4-µm pore) transwell cell culture plates (Costar) were used to separate PECs from naive CD4+ cells. PECs were plated on the superior chamber at a ratio of 1:4 to CD4+ cells, the inferior chamber was coated with anti-CD3/CD28 Abs, and naive CD4+ cells were added. Cultures were maintained for 72 h, then 1 µCi/well [3H]thymidine added (Amersham) and incubated for an additional 18 h. CD4+ cells were transferred to a 96-well plate for harvesting as described.
The role of soluble factors in PEC suppression was assayed by using
varying concentrations (1, 2, and 5 µg/ml) of isotype control or
blocking Abs, anti-IL-10 clone JES5-2A5, anti-IFN- (obtained
from BD PharMingen), and anti-TGF- clone 1D11 (obtained from R&D
Systems), in 96-well plate cocultures, where Abs were added at the same
time as PECs.
Statistical analysis
Data were analyzed with unpaired Student’s t test. Significant values were considered when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies demonstrated that i.p. injections of parasite
Ags, eggs, or filarial parasites led to recruitment of different cell
populations having a variety of activities (9, 15, 16, 17).
Here, we examined the peritoneal cell response in mice after injection
with the glycoconjugate LNnT-Dex at two early time points, 2 and
18 h. The peritoneal cavities of the animals were rinsed with 5 ml
of cold HBSS, and PECs were harvested. Fig. 1
A shows that at both time
points, animals receiving LNnT-Dex rapidly expanded peritoneal cells
compared to animals that received dextran and that at 18 h after
injection, more PECs were recruited to the peritoneal cavity than at
2 h in LNnT-Dex-injected mice.
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b summarizes the main surface markers detected in the
population expanded by LNnT-Dex. The majority of PECs were
Gr1+/CD11b+ (30–40% of total cells; Fig. 1B) and F4/80+/Gr1+ (25–37% of
total cells) compared with Gr1+ (5–7%), CD11b (28% or
higher), and F4/80 (30–50%) in mice injected with Dextran or saline.
In addition, LNnT-Dex-expanded Gr1+ PECs expressed low
levels of B7-2, MHC-II, and CD40, and did not express CD11c, IL-10R, or
NK surface markers. Percentage of composition of PECs from mice
injected with LNnT-Dex or Dextran is provided in the legend for
Fig. 1. PECs expanded by LNnT-Dex exhibit "natural suppressor cell" activity
A number of studies have found that the expanded
Gr1+/CD11b+ cell populations in animals
injected with virus or in mice harboring tumors have suppressive
activity and have called this population of cells "natural
suppressors" (24, 25). Several other studies that used
other pathogens or molecules also have correlated the expression of the
Gr1+ surface marker with suppressive activity
(25, 26, 27, 28). Because LNnT-Dex-expanded PECs also were
Gr1+/CD11b+, we asked whether these cells had
suppressive activity. We tested for suppressive activity of PECs by
initially stimulating naive T cells with anti-CD3 and anti-CD28
Abs and 3 hours later adding PECs from mice injected with LNnT-Dex or
dextran. PECs were added to naive CD4+ T cells at several
ratios. Fig. 2 shows that
CD4+ T cells in the absence of PECs or in the presence of
PECs from saline- or dextran-injected mice exhibited high levels of
proliferation in response to anti-CD3/CD28 stimulation. In
contrast, addition of PECs from LNnT-Dex-injected mice markedly
suppressed proliferation of CD4+ cells, especially when
higher ratios of PECs were added (Fig. 2, A and
B). There was a significant inhibition in the proliferative
response (50% reduction compared to controls) even when the
PEC:CD4+ ratio was 1:8. However, the strongest inhibition
was detected with a PEC:CD4 ratio of 1:4, where greater than 90%
suppression was observed (Fig. 2, A and B).
Suppression by LNnT-Dex-expanded PECs was observed in both BALB/c and
C57BL/6 mice and was characteristic of both time points after LNnT-Dex
injection at which PECs were harvested.
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As shown in Fig. 2, LNnT-Dex-expanded PECs inhibit proliferation
of naive CD4+ T cells in response to anti-CD3/CD28
stimulation. To identify the cell type responsible for this suppressive
activity, we depleted or enriched PECs for Gr1+ cells by
magnetic cell sorting and tested the negative and positive fractions in
PEC:CD4+ cocultures. We found that suppression mediated by
LNnT-Dex PECs was significantly abolished (p <
0.01; Fig. 3a) when
Gr1+ cells were removed (<5%; Fig. 3b) from
the population, indicating that Gr1+ PECs mediate
suppression. In contrast, with PECs enriched for
Gr1+ cells (76% by FACS analysis; Fig. 3B) we observed that the Gr1+-enriched
population exhibited higher levels of inhibition than that observed
with the total PEC population, even when lower PECs:CD4+
ratios were used (Fig. 3A).
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C). This observation suggested that
suppressor activity was attributable to macrophages or neutrophils in
the Gr-1+ fraction (Fig. 3C,
bottom). Suppression is mediated by both cell contact and by soluble factors
We next tested for mechanisms involved in the suppressive activity
of PECs. Initially, we asked if fixed cells would retain suppressor
activity. PECs from LNnT-Dex- or dextran-injected mice were harvested,
adjusted to 5 x 105/ml, and fixed with 0.5%
paraformaldehyde for 5–10 min. PECs then were washed extensively and
added to previously (3 h) stimulated naive CD4+ cells.
Interestingly, fixed PECs from LNnT-Dex-injected mice were able to
partially inhibit the proliferation of anti-CD3/CD28-stimulated
CD4+ cells (Fig. 4A). The level of inhibition
was not as high as that observed in live PECs in cocultures but was
significantly greater than that observed for control fixed PECs
(p < 0.05), demonstrating that cell-to-cell
interactions are partially responsible for the observed suppression. We
next addressed the possibility that soluble factors also may
participate in PEC-mediated suppression. In these experiments,
transwells were used to eliminate cell-to-cell contact in
PEC:CD4+ cocultures. PECs were plated in the upper chamber
at a ratio of 1:4 with respect to naive CD4+ cells, which
were plated and stimulated in the lower chamber with anti-CD3/CD28
plate-bound Abs. The transwell cocultures showed that even in the
absence of cell-to-cell contact, there was significant inhibition in
the proliferation of anti-CD3/CD28-stimulated CD4+
cells (
50% inhibition; Fig. 4B; p <
0.05), suggesting a role for soluble factors in Gr1+
PEC-mediated suppression of T cell proliferation.
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To determine if LNnT-Dex injection biased as well as activated
PECs, we examined the spontaneous cytokine profiles from peritoneal
cells at 2 h or 18 h after injection of LNnT-Dex or dextran
in Th1-type (C57BL/6) and Th2-type (BALB/c) mice. After 72 h of
culture, supernatants were harvested and analyzed. The results revealed
that injection of LNnT-Dex profoundly altered the pattern of cytokines
spontaneously released by PECs compared to PECs from dextran-injected
mice, regardless of the strain. PECs expanded by LNnT-Dex produced
significantly lower levels of the proinflammatory cytokines IL-1,
IL-12, IL-18, and IFN-, and in contrast, higher levels of IL-10 and
TGF- than PECs from dextran-injected mice (Tables I and II). We did not detect significant
levels of PGE2 or nitric
oxide from either population of PECs (data not shown). Thus, LNnT-Dex
not only expands Gr1+/F4/80+ suppressor
cells, but activates these cells towards an antiinflammatory cytokine
profile in both Th1- and Th2-type strains of mice.
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production in cocultures
with naive CD4+ T cells while enhancing the production of
IL-13
Previous studies on natural suppressor cells suggested that after
interaction with suppressors, CD4 or CD8 target cells undergo apoptosis
(24, 25, 26). To exclude the possibility that CD4+
cells in our cocultures had reduced proliferation attributable to cell
death, we measured cytokine production in response to anti-CD3/CD28
stimulation in the presence of PECs. We found that there was a
significant decrease in IFN- production in supernatants from
cocultures containing PECs expanded by LNnT-Dex compared to PECs from
dextran-injected or noninjected mice (Fig. 5A; p <
0.05). Additionally, we observed that levels of IL-13 were
significantly elevated in cocultures with LNnT-Dex-expanded PECs
compared to cultures containing dextran-recruited PECs (Fig. 5B; p < 0.01). Furthermore, IL-4 levels
were increased from 587 ± 130 pg/ml to 1227 ± 337 pg/ml in
cocultures containing LNnT-Dex-expanded PECs. These results demonstrate
that cocultured CD4+ cells are alive and that PECs expanded
by LNnT-Dex can alter the cytokine profile of naive CD4+ T
cells.
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We found that PEC-mediated suppression of naive CD4+ T
cells could be reversed by removal of Gr1+ PECs, and
further, that cocultured CD4+ T cells were viable and
responsive when PECs were removed. Secondly, we found that supernatants
from cocultures were biased in a Th2-type, anti-inflammatory mode.
Therefore, we asked if cocultured CD4+ cells would respond
to secondary stimulation and whether initial culture with
Gr1+ suppressor cells "imprinted" a Th1- or Th2-type
response on the CD4+ T cells. After 72 h of coculture,
cells were removed, washed, and CD4+ cells repurified.
Purified CD4+ cells then were plated and incubated for
72 h (rested), and then stimulated with anti-CD3/CD28
plate-bound Abs in the absence of PECs. Supernatants were harvested
24 h later. Similar to what was detected in the primary coculture
supernatants, we found that rested and restimulated CD4 cells produced
low levels of IFN- (Fig. 6A) and in contrast, produced
high levels of IL-13 (Fig. 6B). These data provide
additional evidence that CD4+ cells in cocultures are
alive, and furthermore, suggest that the majority of T cells isolated
after primary coculture with LNnT-Dex-PECs are Th2 committed, as the
highest levels of IL-13 were secreted after secondary stimulation
(29). In parallel experiments, we found that the
addition of IL-12 to cocultures partially restored IFN- production
as detected in supernatants, providing further evidence that cocultured
CD4+ cells are alive and able to respond to external
stimuli (data not shown). In addition, exogenous IL-12 in cocultures
also reduced the levels of IL-13. Although this suggests that these
cells have an imprinted Th2-like phenotype, additional and more
detailed experiments need to be performed to verify this important
observation.
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, partially abrogates
suppression activity in PECs recruited by LNnT-Dex
Because a significant inhibitory effect was shown in experiments
where PECs:CD4+ cocultures were performed with transwells
and because LNnT-Dex PECs produced high levels of IL-10 and TGF-, we
performed neutralization experiments to determine whether either of
these molecules classically associated with inhibitory effects
(30, 31, 32, 33) were involved in suppression. As shown in Fig. 7, neutralization of IL-10 resulted in a
partial decrease (30%; p < 0.05) in the ability of
the LNnT-Dex PECs to suppress CD4+ cell proliferation. In
contrast, neutralizing Abs against TGF-1, TGF-2, and TGF-3 had
no significant effect on cellular proliferation (Fig. 7). This suggests
that IL-10 plays a modest role as a soluble factor in the suppressive
activity associated with LNnT-Dex-expanded PECs.
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and NO abrogates suppressive activity in PECs
expanded by LNnT-Dex
The majority of studies on Gr1 suppressors have shown that
suppression is an IFN- and NO-dependent process (30, 34, 35). Because blocking IL-10 in cocultures was unable to
completely restore proliferative activity in CD4+ cells, we
asked if LNnT-Dex-expanded Gr1 suppressors functioned in an IFN--
and NO-dependent mechanism. As shown in Fig. 7, both the neutralization
of IFN- and the inhibition of NO via
N-monomethyl-L-arginine completely
restored the proliferative response of CD4+ cells. Taken
together with the partial reversal of suppression by neutralizing
IL-10, it appears that LNnT-Dex-expanded Gr1 suppressors function via
several factors to suppress naive CD4+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we observed that Gr1+ suppressor cells were rapidly expanded in the peritoneal cavities of naive mice after a single injection of the glycoconjugate LNnT-Dex, of which LNnT is found in human milk and on helminth parasites. Injection of LNnT-Dex into naive mice expanded Gr1+/CD11b+/F4/80+ cells as early as 2 h after injection compared to dextran- or saline-injected controls. This rapid expansion of Gr1 suppressors has not been reported in previous studies on natural suppressors; rather, these other reports showed that Gr1+ suppressor expansion took from days to weeks in animals harboring parasites or tumors or exposed to virus or viral antigens (24, 25, 28, 35). In addition, we noted that this population was maintained in vivo for at least 24 h after injection of LNnT-Dex. The rapid increase in frequency of Gr1+ suppressors by LNnT-Dex suggests that the glycoconjugate functions via an innate immune response.
LNnT-Dex-expanded PECs were functional suppressors, as they adoptively suppressed a primary, naive CD4+ T cell response to anti-CD3 and anti-CD28 Abs. Suppression was shown to be dependent on the presence of Gr1+ cells, as removal of Gr1+ cells from the PEC population rendered the remaining PECs unable to suppress CD4+ cell proliferation, even when cocultured at a 1:2 ratio of PECs:CD4+ cell. In contrast, the Gr1+-enriched cell fraction maintained strong suppressive activity, confirming a suppressor role for cells carrying this marker. When performing cocultures to test suppressor activity of PECs, we used a ratio of PECs:CD4+ T cells that was similar/identical to those used in other studies where tumors or virus were used to expand Gr1+ suppressors (24, 25, 28, 34, 35). These ratios are in contrast to the only other study examining suppressor PECs generated by parasitic helminths. In that study, the ratio of PECs to target cells was 10- to 100-fold higher than the ratio used in our study (15).
Mechanistically, we demonstrated that Gr1+ suppressor cells
functioned via both cell-to-cell contact and through the release of
soluble factors. Thus, although paraformaldehyde-fixed PECs did not
suppress naive CD4+ cells at the same level as nonfixed
PECs, they did reduce CD4+ T cell proliferation well below
the level seen with nonfixed PECs from dextran-injected mice. This
finding indicates that a molecule(s) associated with PEC membranes is
involved in suppression, and furthermore, that they are rapidly
up-regulated on the cell surface after interaction with LNnT-Dex.
However, cell contact alone was not sufficient for optimal suppression,
and our transwell experiments demonstrated a role for soluble factors,
where IL-10, IFN-, and NO appear to be involved, but not TGF-.
The soluble factors implicated in our study are similar to those seen
in other suppressor cell populations induced by pathogens that have
been shown to be contact-, IFN-- and NO-dependent (26, 27, 34). Of note, the PECs expanded by LNnT-Dex or dextran did not
spontaneously release NO in culture (data not shown); however, when
cultured with IFN- producing CD4+ cells, they release
NO. Release of NO by macrophages or myeloid cells following cell
contact is a process normally mediated by CD40 ligation
(34). Although we cannot rule out a role for CD40 here,
FACScan analysis showed that CD40 was expressed at low levels on
LNnT-Dex-expanded Gr1+ suppressor cells. Perhaps the
strongest argument against a required role for CD40 are our preliminary
studies in CD40 knockout mice, which show that LNnT-Dex expands
functional Gr1+ suppressors in these mice (data not shown).
Finally, in regards to mechanisms of suppression, the majority of other
studies have shown that the target population
(CD4+/CD8+) undergoes apoptosis after contact
with Gr1+ suppressors (24, 25, 26, 27, 32). In
contrast, we found that CD4+ cells were viable after
coculture with Gr1+ suppressors, and that after rest and
restimulation, supernatants obtained from cocultures contained high
levels of cytokines, indicating that the cells were alive. One of the
more striking observations from the coculture experiments was that
rested and restimulated CD4+ T cells maintained the profile
of cytokine production seen in the initial cocultures, which was low
levels of IFN- and high levels of IL-13 compared with their
respective controls. Taken together, these data support the fact that
CD4+ cells remain viable in the presence of PECs recruited
by LNnT-Dex and that the Gr1+ suppressor cells may
"imprint" a Th2 phenotype on naive CD4+ cells.
Though our results differed in both the time required to expand
Gr1+ suppressors as well as the ratio of
PECs:CD4+ cells used in cocultures, our results on
mechanism of suppression are in accordance with the findings of
MacDonald et al. (16) and Loke et al. (15)
who implanted the nematode Brugia malayi in the peritoneal
cavity of mice to expand suppressors macrophages. Loke et al.
(15) also showed that cytokine production in their system
was skewed towards Th2-type concomitant with suppression in the
proliferative response (36). These studies suggest that
the commitment towards a Th2 pattern of cytokine production by
CD4+ cells after interaction with PEC suppressors may
explain the reduced levels of IFN- and IL-12 observed in certain
helminth infections.
The mechanism used by the granulocyte-monocyte lineage to
differentially inhibit the function of CD4+ T cells is not
yet well understood (34, 35). We currently are
investigating several hypotheses on suppressive mechanism and
imprinting of Th2-type phenotype on naive T cells in coculture.
Although we were able to show that soluble factors such as IL-10,
IFN-, and NO were involved in suppression, we were surprised to find
that blockade of TGF-, a cytokine known to induce suppression in
activated T cells (31, 38) did not restore
CD4+ proliferation. However, the suppressive effect of
TGF- is not restricted to inhibition of proliferative responses, but
in addition, it has the ability to modulate the profile of activating
cytokines, such as limiting the production of IFN- (31, 38). In our system, the LNnT-Dex-induced PECs produced high
levels of TGF-, which may have been partly responsible for the
reduced levels of IFN-. In contrast to TGF-, IL-10 did have a
small effect in suppression as shown in the neutralizing Ab
experiments. The ability of parasite-expressed glycoconjugates to
induce production of IL-10 and TGF- may be a major mechanism used by
the parasite to avoid both a strong and an early cellular immune
response during infection. This correlates with our results that showed
that the helminth- and human milk-expressed sugar LNFPIII could be
added in vitro to down-modulate IL-10 production in PBMCs from patients
infected with S. mansoni (39). Taken together,
PECs expanded by the immuno-modulatory glycoconjugate LNnT-Dex produce
two key cytokines with suppressive or anti-inflammatory activity, IL-10
and TGF-, which are capable of counterbalancing the proinflammatory
cytokines IL-1, IL-12, IL-18, and IFN- (30, 40, 41).
As both LNnT and LNFPIII expand suppressor cells and drive IL-10
production and are also found in human milk, which is
immunosuppressive, we speculate that the immunosuppressive properties
of human milk are attributable in part to the presence of these two
glycoconjugates (23).
The rapid and local responses to the LNnT-Dex glycoconjugate that we have shown suggests that these sugars may function therapeutically when an effective and fast anti-inflammatory response is needed, or as a potential adjuvant to induce Th2 responses later in disease progression. The precise nature of cells responsible for the suppressive effect reported here is presently under further investigation. We already have shown that a Gr1+ population is directly related to suppression. Furthermore, low or absent expression of MHC-class II, CD40, and B7-2 detected in the Gr1+ population suggest an immature myeloid cell type (36). A similar cell population has been found from bone marrow that induces hyporesponsiveness in allogenic T cells (34) and immune dysfunction in cancer (37).
Collectively, we presented data demonstrating that an immunomodulatory
glycoconjugate (LNnT-Dex) comprised of a tetrasaccharide found on
helminth parasites and in human milk rapidly expands a suppressor cell
population that, similar to previous reports, mediates suppression via
an IFN-- and NO-dependent mechanism, but unlike other reports, also
may use IL-10. The biological significance of suppressor cells, which
are expanded by parasite-expressed carbohydrates in helminth and other
infections, remains to be investigated. It is known that immunodominant
epitopes found on glycoproteins and glycolipids are often periodate
sensitive (42, 45), implicating a role for glycosylated
epitopes in the host-parasite interaction (46, 47). In
light of our observations, we suggest that these immunoregulatory
oligosaccharides found in milk and on helminth-expressed
glycoconjugates may have additional roles in driving early
immunological events towards Th2-type or antiinflammatory responses,
and furthermore, that these molecules represent novel potential
therapeutic agents for the treatment of Th1-mediated autoimmune
diseases as well as any type of proinflammatory condition.
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Footnotes |
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2 Abbreviations used in this paper: LNFPIII, lacto-N-fucopentaose; LNnT, lacto-N-neotetraose; LNnT-Dex, LNnT-dextran; PEC, peritoneal exudate cells.
Received for publication March 8, 2001. Accepted for publication August 22, 2001.
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