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Erratum |
*
Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh, U.K.;
Department of Biology, Imperial College of Science, Technology and Medicine, London; and
The Rayne Laboratory, Medical School, University of Edinburgh, Edinburgh, United Kingdom.
Abstract
Loss of T lymphocyte proliferation and the emergence of a host
response that is dominated by a Th2-type profile are well-established
features of human filariasis. We have previously reported that adherent
peritoneal exudate cells (PEC) from mice transplanted with adult
Brugia malayi parasites suppress the proliferation of
lymphocytes without blocking Ag-cytokine production in vitro. We now
show that infection of mice with the infective larval (L3) stage of
B. malayi generates a similar population of PEC.
Suppressive cells are generated within 7 days of infection and mediate
their effects through a nitric oxide-independent pathway. Both L3 and
adult infection elicit high levels of host IL-4 whereas the
microfilarial stage of the parasite induces IFN-
production and does
not generate a similar form of suppression. Production of host IL-4 was
necessary to allow the generation of suppressive PEC, given that
IL-4-deficient mice implanted with adult parasites failed to induce
proliferative block. However, IL-10-deficient mice implanted with adult
parasites resulted in T cell suppression, indicating that IL-10 is not
essential for the induction of hyporesponsiveness. Neither IL-4 nor
IL-10 were directly responsible for ablating cellular proliferation in
vitro, as the addition of neutralizing Ab to either cytokine did not
reverse the proliferative block. Thus, IL-4 produced in vivo in
response to filarial L3 and adult parasites is essential for the
induction of proliferative suppression but is not itself the
suppressive factor.
The establishment of a parasitic helminth infection is characteristically accompanied by a host response dominated by a Th2-type profile and the emergence of parasite-specific immunosuppression (1, 2, 3, 4). This is the case in human filariasis, in which long term infection with this lymphatic-dwelling nematode is accompanied by down-regulated host immune responses (5, 6, 7). However, the mechanisms that underlie the development of the profound cellular hyporesponsiveness in filariasis and the relationship of this to the development of a Th2-type response are unknown. These questions can be addressed in Brugia malayi-infected mice, because Th subset bias and immune suppression, both specific and nonspecific, are hallmarks of infection with this filarial nematode (8, 9, 10, 11).
We investigated the possibility that filariae down-regulate host immune responses by interfering with effective APC function. To delineate the role of the APC, we tested their competence in a defined model Ag system. Mice were implanted in the peritoneal cavity with adult B. malayi, and the ability of adherent peritoneal cells to present Ag ex vivo was evaluated (11). We found that when live but not dead parasites were used, the ability of the D10.G4 Th2 cell clone to proliferate in response to its cognate Ag (conalbumin) was completely blocked. However, levels of Ag-cytokine production were intact, and even elevated, relative to control peritoneal exudate cells (PEC),3 indicating that there was no defect in Ag processing. When low numbers of infection-derived PEC were mixed with an excess of normal irradiated splenocytes, profound proliferative suppression was still observed. This demonstrated that suppression was an active process and not due to an absence of costimulatory molecules or reduced numbers of effective APC. Moreover, while suppression could be transferred with supernatants from cultured PEC, this was not due to soluble factors typically associated with "suppressor macrophages" such as prostaglandins or nitric oxide (11).
In contrast to adult B. malayi, peritoneal implant of the
blood-circulating microfilarial stage (Mf) of the parasite generates a
less profound form of suppression that is reversible by addition of NO
inhibitors in vitro (11). Previous studies have demonstrated that
contrasting cytokine profiles are induced by these two stages of the
parasite. Implantation of adult parasites induces high levels of IL-4
with a matching "Th2" Ab isotype profile, while microfilariae
induce IFN- and a "Th1" Ab profile (10). In this study, we chose
first to evaluate the infective larval stage (L3) which, like the adult
stage, induces high levels of IL-4 in the infected host (9, 12) and, as
the stage of parasite that establishes infection, must also have means
to avoid host immune responsiveness. A striking correlation was
observed between splenic IL-4 production and the level of proliferative
suppression in L3-infected animals. We therefore investigated the role
of IL-4 in the in vivo induction of proliferative suppression. This
work has led to the identification of a potentially novel form of
immunosuppression induced by filarial infection that is dependent on
IL-4 but not the down-regulatory cytokine IL-10.
Materials and Methods
Parasite material
B. malayi adults and Mf were obtained from infected jirds purchased from TRS laboratories (Athens, GA). Adult worms were removed from the peritoneal cavity and washed in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 50 µg/ml gentamicin. L3 were obtained from Aedes aegypti mosquitoes that had been fed on blood containing Mf 12 days previously. Adult B. malayi Ag (BmA) was prepared by homogenization of mixed sex worms in PBS on ice followed by centrifugation at 10,000 x g for 20 min. The resultant supernatant was passed through a 0.2-µm pore size filter before protein concentration determination by the Coomassie Plus protein assay (Pierce Chemical Co., Rockford, IL).
Mouse infection model
For all experiments, mice used were 6- to 8-wk-old males unless otherwise stated. CBA/Ca mice plus age-matched control mice were purchased from Harlan-U.K. (Bicester, U.K.). IL-4-deficient (IL-4-/-) (13) C57BL/6, IL-10-deficient (IL-10-/-) (14) C57BL/6, and wild-type C57BL/6 mice were purchased from B&K Universal Ltd. (North Humberside, U.K.) with the permission of the Institute of Genetics, University of Cologne, Cologne, Germany. Mice were implanted i.p. with either 6 live adult B. malayi females or 10 to 400 L3 (as shown in text). Implants were left for 3 wk unless otherwise stated. At this point, mice were euthanized by cardiac puncture, and PEC were harvested by thorough washing of the peritoneal cavity with 15 ml of RPMI containing 50 µg/ml gentamicin.
For treatment of animals with neutralizing Ab to IL-4 in vivo, mice
were injected i.p. with 2 mg of anti-IL-4 mAb (11B11), or
isotype-matched anti-IFN- mAb (R4-6A2; American Type Culture
Collection (ATCC) HB170) at the time of parasite implant. For in vivo
use, 11B11 was kindly provided by F.W. Falkenberg (Ruhr University,
Bochum, Germany), and for an isotype-matched control, the miniPERM mAb
production system (Heraeus) was used to produce neutralizing mAb to
IFN-, purified using a protein G column (Pharmacia Biotech, Uppsala,
Sweden).
Cultures and cell lines
All in vitro cultures were conducted in RPMI 1640 medium (Life Technologies) supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µM 2-ME, and 10% FCS (complete medium). The Th2 cell clone D10.G4 (15) and the B cell hybridoma HB32 (ATCC, Rockville, MD) were maintained in culture as previously described (11). For experiments with C57BL/6 mice, primary OVA-sensitized T cell lines were established by s.c. immunization of mice with 50 µg/footpad OVA in CFA. Two weeks later, popliteal lymph nodes were removed, and liberated cells were cultured in complete medium with 25 µg/ml OVA. After 3 to 4 days, cultures were supplemented with 10% (v/v) conditioned medium from a 48-h culture of conalbumin-stimulated mouse lymphocytes. Syngeneic splenocytes inactivated by irradiation with 2000 rads were added at the same time as feeders. T cells were used 5 to 7 days after this rest period.
Proliferation and cytokine assays
For investigation of proliferative suppression caused by parasite-exposed PEC, 100 µl of PEC at 1 x 106/ml were adhered to a flat-bottom 96-well plate (Nunclon; Life Technologies) at 37°C for 2 to 3 h, after which nonadherent cells and Mf were removed by washing with complete medium. Plates with adherent PEC were then used for coculture with 5 x 104 D10.G4 or HB32 cells or with primary T cell lines, to a final volume of 200 µl/well. Conalbumin and OVA (both from Sigma Chemical Co., St. Louis, MO) were used at the concentrations indicated in the text. After incubation for 48 h at 37°C, 100 µl of supernatant were removed from each well of the D10.G4 or primary lymphocyte assays for cytokine analysis; 1 µCi of [3H]TdR in 10 µl of complete medium was then added to each well, and plates were incubated for 16 to 18 h at 37°C before harvesting and counting using a Top Count microplate scintillation counter (Packard Instrument Co., Meriden, CT).
For analysis of lymphocyte responses in the spleens of infected animals, splenocytes were obtained by teasing spleens apart and then lysing erythrocytes using red blood cell lysis buffer (Sigma Chemical Co.). Viable cells were then counted by trypan blue exclusion. Whole spleen cells were incubated at 5 x 105 cells/well with BmA at a final concentration of 5 µg/ml. After incubation for 65 h at 37°C, supernatants were taken for subsequent cytokine analysis.
The IL-2/IL-4-responsive NK cell line (16) was used to measure cytokine
production by D10.G4 cells, primary T cells and splenocytes, as
previously described (10, 11). Briefly, proliferation of the NK cells
at 104 cells/well was measured in the presence of 20 µl
of culture supernatant with the addition of anti-IL-2 (S4B6)
neutralizing Ab for IL-4 measurement. S4B6 was obtained from ascites
for in vitro use and used at 2.5 µl/ml, the optimal concentration for
neutralization as determined by titration. Cells and supernatants were
incubated for 24 h at 37°C before addition of 1 µCi of
[3H]TdR in 10 µl of complete medium. After a further
12 h of incubation at 37°C, plates were harvested and counted.
IFN- was measured by ELISA using R46A2 (ATCC) as capture Ab and
biotinylated rat anti-mouse IFN- monoclonal XMG1.2 Ab
(PharMingen, San Diego, CA, 18112D), followed by avidin-alkaline
phosphatase (Sigma Chemical Co., A-7294) for detection. Standard curves
using mouse recombinant IL-4 (Sigma Chemical Co., I-1020), and IFN-
(Sigma Chemical Co., I-5517) were performed to determine cytokine
levels in supernatants.
Neutralizing Ab to mouse IL-10 was purchased from Genzyme Diagnostics (Cambridge, U.K.) and used at 2 µg/ml. The neutralizing Ab to IL-4 (11B11) was obtained from ascites for in vitro use and used at 2.5 µl/ml, the optimal concentration for neutralization as determined by titration. The NO inhibitor L-NG-monomethylarginine (L-NMMA) and the control inhibitor D-NMMA (Wellcome Foundation, Beckenham, U.K.) were used where indicated at a final concentration of 250 µg/ml.
Cell sorting and analysis
PEC isolated from mice were washed and adjusted to 1 x 107/ml in PBS containing 0.02% EDTA. Cells were then incubated at 4°C for 30 min with the appropriate concentration of anti-macrophage (Clone F4/80, R-phycoerythrin-conjugated, Caltag Laboratories, San Francisco, CA) mAb, as determined by titration. F4/80-positive and -negative PEC were sorted using a FACStar (Becton Dickinson, San Jose, CA), with logarithmic amplification of fluorescence detection and side scatter and linear amplification of forward scatter. LYSIS 2 software was used for acquisition and analysis. Sorted cells were then washed and resuspended in complete RPMI medium for use in the D10.G4 assay.
Cytocentrifuge preparations of PEC (1 x 105 cells/slide) were air-dried and fixed in methanol before staining with Diff-Quik (Dade, Diagnostics, Unterschleissheim, Germany). The proportion of macrophages, lymphocytes, mast cells, and eosinophils was determined by morphologic examination of at least 300 cells in randomly selected fields using an Olympus BH2 microscope with a x100 objective.
Statistical analysis
Student’s t test and the paired t test were used to determine the statistical significance of differences between and within groups. P < 0.05 was considered to be a significant difference.
Results
Time course of suppression by adult B. malayi
In previous work, we demonstrated that 3 wk after adult B.
malayi implantation into mice, a suppressive cell population was
recovered from the peritoneal cavity. Because L3 stage parasites do not
survive for 3 wk in the murine host (17), a time course experiment was
conducted to establish a shorter time frame, as a prelude to testing L3
themselves. Following implantation of adult B. malayi,
proliferation of the murine T cell clone D10.G4 cultured with adherent
peritoneal cells from infected mice was measured at several time points
during a 3-wk period (Fig. 1
A). Seven days of host
exposure to the parasite were required to achieve significant
proliferative suppression (P < 0.05). This
suggests that the development of a host immune response or recruitment
of particular cells is required, consistent with the inability of
parasites to directly induce proliferative suppression in vitro
(11).
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B). In this case, suppression of proliferation was
apparent with cells taken 7 days postexposure to adult parasites but
was maximal with day 14 cells. We thus chose to test cells from
L3-infected mice at 2 wk postinfection to ensure that sufficient time
had elapsed for the development of a fully suppressive cell
population. Infective stage larvae and adult parasites induce similar profiles of T cell suppression in infected animals
A preliminary experiment showed that 600 L3 parasites implanted
i.p. into CBA/Ca mice generated a population of adherent peritoneal
cells which induced profound and significant cellular proliferative
suppression (P < 0.04) in cultured T cells that
was not reversible with the NO inhibitor L-NMMA (Table I). We have previously reported similar
findings in adult parasite-implanted mice, because even at minimal PEC
numbers mixed with a 10-fold higher concentration of control APC, adult
parasite-derived suppression cannot be reversed by NO inhibitors
(11).
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A). At high numbers of
implanted L3 (400), suppression was greater than that seen with adult
implants (P < 0.05), despite the lower biomass
of 400 L3s compared with 6 adult parasites. Suppression was not
reversed at any parasite dose by L-NMMA (data not
shown).
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Analysis of Ag-IL-4 production by D10.G4 cells showed that
although cellular proliferation was ablated in the presence of both
adult and L3 implant-derived PEC, cytokine production was enhanced
(Fig. 2B). This argues against the possibility that
the reduced proliferation of the D10.G4 cells was due to
ineffective Ag presentation, reduced numbers of APC, or heightened cell
death.
In addition to T cells, proliferation of the B cell hybridoma HB32 was
assessed in the presence of adherent PEC from L3-implanted animals.
A significant reduction in hybridoma cell proliferation was seen with
administration of as few as 10 L3 parasites (Fig. 2C)
(P < 0.02).
Consistent with previously published data, splenocytes taken from
L3-implanted animals produced elevated IL-4 levels (Fig. 3A) in response to
parasite Ag (12). Increasing levels of splenocyte
Brugia-specific IL-4 production correlated with increasing
size of L3 inoculum and inversely correlated with suppression. IFN-
levels were also elevated in L3-implanted animals, although there was
no correlation between cytokine levels detected and inoculum dose (Fig. 3B).
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Because of the correlation between splenic IL-4 production and the
generation of PEC that block proliferation, we chose to investigate the
hypothesis that IL-4 plays a role in the development of the suppressive
PEC, by the administration of neutralizing Ab to IL-4 (11B11) at the
time of parasite implant. Adult parasite-implanted mice that received 2
mg of anti-IL-4 at the time of implant had impaired ability to
generate strongly suppressive PEC. In contrast, PEC from control
implanted mice, or implanted mice injected with isotype-matched
anti-IFN-, significantly blocked cellular proliferation (Fig. 4A)
(P < 0.01). This effect was achieved under
conditions in which anti-IL-4 did not block the subsequent
development of IL-4-producing T cells in the spleens of implanted mice
(Fig. 4B). Thus, a reduction in IL-4 levels at the
time of initial exposure to the parasite was sufficient to reduce the
ability of PEC to suppress proliferation.
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To more firmly establish the role of IL-4 in the induction
of PEC suppression, mice genetically deficient for the production of
IL-4 (IL-4-/-) were implanted with adult B.
malayi parasites. The ability to stimulate Ag proliferation was
compared in PEC taken from knockout and wild-type C57BL/6 animals
implanted with B. malayi. Cellular proliferation as measured
by [3H]TdR incorporation showed that in contrast to
wild-type mice, IL-4-/- mice implanted i.p. with adult
B. malayi parasites failed to generate adherent PEC that
blocked the proliferation of either OVA-specific T cells (Fig. 5A) or the B cell
hybridoma, HB32 (Fig. 5B). Assessment of splenocyte
cytokine responses showed that adult parasite-implanted
IL-4+/+ mice produced elevated levels of parasite-specific
IL-4 relative to uninfected controls. As expected, no IL-4 was produced
by IL-4-/- mice (data not shown).
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Because PEC from IL-4+/+ mice suppress
proliferation of the IL-4-secreting clone D10.G4, it is unlikely that
IL-4 itself can be the effector molecule for blocking proliferation.
This was confirmed by the inability of 11B11 to reverse the
proliferative block in vitro of either the D10.G4 clone or the HB32
hybridoma (Table II). We also tested
IL-10 because it exerts major down-regulatory effects in vitro in
parasitic helminth infection (18) and in the development of
tumor-derived suppression (19). Addition of neutralizing Ab to IL-10
did not restore proliferative responses of T cells exposed to infection
derived PEC (Table II). The removal of IL-10 did enhance responses with
control-derived PEC, with increased proliferation of D10.G4 cells seen
in these cultures.
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Having established that in vivo production of IL-4 is necessary
for the induction of competent suppressor cells by filarial implant,
and given the importance of IL-10 as a down-regulatory cytokine
influenced by IL-4, we decided to investigate the role of IL-10 in vivo
in the induction phase of suppression. C57BL/6 mice deficient in the
production of IL-10 (IL-10-/-) were implanted with
adult B. malayi parasites. Adherent peritoneal exudate cells
taken from these mice were capable of inducing proliferative
suppression in OVA-specific T cells as effectively as wild-type mice
(Fig. 6A), thus IL-10
is not required for either the induction or effector phases of T cell
hyporesponsiveness. However, suppression of proliferation of the B cell
hybridoma HB32 was not as complete as that seen in similarly infected
IL-10+/+ mice (Fig. 6B), suggesting that
the suppressive PEC population may encompass a minor IL-10-dependent
pathway effective on B cells. Similar levels of IL-4 were produced by
splenocytes from IL-10+/+ and IL-10-/-
infected mice, with both groups producing much greater levels of
cytokine than uninfected animals in response to parasite Ag challenge
in vitro (data not shown).
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To investigate the dynamics of cell recruitment during infection,
the percentage of different peritoneal cell subpopulations was
determined from cytocentrifuge preparations taken from control mice or
mice implanted with Brugia adults or L3. Parasite-implanted
mice had a marked reduction in mast cells and an increase in both
macrophages and eosinophils, compared with unimplanted control mice
(Fig. 7). Recruitment of eosinophils to
the peritoneal cavity was seen in mice implanted with as few as 10 L3
parasites. Parasite-implanted mice showed a dramatic increase in total
cell numbers recruited to the peritoneal cavity in comparison to
unimplanted control mice; three to five times as many cells were
routinely recovered from implanted animals. Thus, increased ratios of
macrophages and eosinophils reflect not only a proportional increase
but also a dramatic increase in total cell numbers of each type. In
contrast, despite the increased number of cells in parasite-implanted
mice, both the percentage and total number of mast cells were
decreased.
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Given the well-documented role of macrophages in cellular
suppression (20, 21), the expansion of macrophage numbers in the
peritoneal cavity of parasite-implanted mice (Fig. 7), and the adherent
properties of the suppressive cell, we investigated the possibility
that macrophages were responsible for proliferative block. Peritoneal
cells from 10 control or adult parasite-implanted mice were combined
and sorted by FACS into macrophage positive or negative populations
using the macrophage-specific mAb F4/80. Greater than 97% purity was
achieved for each group (data not shown). Proliferation of the T cell
clone D10.G4 was then measured in the presence of each cell population
(Fig. 8). D10.G4 cells proliferated well
when cocultured with the macrophage-enriched population, while
F4/80- cells prevented cellular proliferation. The
absence of proliferation in the F4/80- cultures was not
due to a lack of effective APC, because high levels of Ag-IL-4 was
produced in these cultures (data not shown).
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Parasite immune evasion strategies are increasingly being characterized in terms of host cytokine networks (1, 22, 23). In the case of the filarial nematode B. malayi, as with most other helminth parasites, infection stimulates a dramatic Th2-type response with abundant IL-4 production whether exposure occurs to the L3 or adult stage of the parasite (10, 25). A central question, therefore, is whether the characteristic Th2-type response is a necessary prerequisite for the induction of suppression in filarial infection. The data presented herein demonstrate that host IL-4 production is indeed essential for the induction of a nonspecific suppressor cell population but that the major down-regulatory cytokine associated with Th2 responses, IL-10, is not a key component.
IL-4 production is likely to be one of the first events to occur following exposure to helminth parasites (24). Production of IL-4 is greatly expanded in Brugia infected mice (9, 10, 12), and in this study we see a direct correlation between parasite-specific splenocyte IL-4 production and PEC-mediated suppression of lymphocyte proliferation at each dose of L3. IL-4-/- mice were unable to generate a suppressive PEC population, formally demonstrating that IL-4 is required. We cannot yet determine whether IL-4 alone is sufficient for suppressive PEC development or whether a mature Th2 response must first emerge. However, mice given anti-IL-4 at the time of implantation still develop "Th2" responses but are unable to generate full suppression. This suggests that it is early IL-4 production, and not necessarily Th2 establishment, that may be the essential factor in the development of a proliferative block.
The ablation of cellular proliferation in this system is due to
soluble factor(s) released from host PEC that does not include IFN-,
nitric oxide, or prostaglandins (11). A direct role in vitro for IL-4
has been implicated in the L3-induced down-regulation of IL-2-driven T
cell mitogenic proliferative responses (25). However, IL-4 is not the
PEC-derived suppressive factor, as proliferative responses are not
restored by the addition of neutralizing anti-IL-4 Ab in vitro, and
the Th2 cell clone D10.G4 itself produces copious amounts of IL-4
during its normal proliferative response.
IL-10 has been implicated as a mediator of filarial nonproliferative responses in human patients in some (26, 27) but not all (28) studies. Surprisingly, in our experiments neutralization of IL-10 in vitro did not restore proliferation to treated cultures, and IL-10-/- mice generated full suppression of the T cell clone D10.G4. However, a partial role for this potent down-regulatory cytokine is suggested by the fact that, in contrast to the wild-type, IL-10-/- mouse implant-derived PEC induce less profound suppression of hybridomas.
TGF-ß is another cytokine with down-regulatory ability that has been reported to reduce macrophage antiparasitic responses during helminth infection (29). Moreover, TGF-ß, together with IL-10, has a close relationship in the development of tumor-related immunosuppression (19). TGF-ß appears to be a likely candidate since we have previously shown that at very low concentrations of parasite-exposed cells, enhanced proliferation can be seen (11), consistent with the reported properties of TGF-ß (30). However, addition of two commercially available anti-TGF-ß Abs did not restore proliferation to suppressed cultures in vitro, either alone or in combination with Ab to IL-10 (11) (data not shown). Nonetheless, TGF-ß remains an attractive possibility that is consistent with the data, and we are still investigating this cytokine as a potential mediator. Other potential candidates remain to be investigated, including such possibilities as glycosphingolipids (31) and other nonpeptide mediators. However, given the profound nature of the suppression and its IL-4 dependence, we are considering the possibility that the proliferative block is effected by a suppressive factor not previously described, possibly acting in combination with known down-regulatory mechanisms.
We have previously shown that Brugia-specific T cells generated in the peritoneal cavity fail to proliferate in response to parasite Ag while producing IL-2 and IL-4, but that this proliferative block is not Ag specific given that Con A proliferative responses are also ablated (11). The induction of nonspecific host antiproliferative mechanisms does not currently account for the Ag-specific hyporesponsiveness observed during human filarial infection. Although some evidence for generalized unresponsiveness in human filarial infection is found in onchocerciasis (32), studies on lymphatic filariasis generally show that only parasite-specific proliferation is lost (5, 6, 33). However, animal studies have demonstrated that both specific and nonspecific proliferative suppression can be induced by infection with Brugia species (11, 34, 35, 36, 37). The possibility that a cell population that is profoundly antiproliferative may lead to the development of Ag-specific cell suppression remains to be explored. For example, if naive T cells first encounter Ag in an environment in which they are unable to proliferate, this may render them tolerant or anergic to later antigenic challenge (38).
Alternatively, the suppression we describe here may be important only at the site of infection, where potentially damaging host inflammatory responses in the local environment are down-regulated. Adult worms live within the lymphatic vessels for many years, apparently impervious to damage by the host immune system (39). We have found that adult excretory-secretory (ES) material injected daily into mice can induce the PEC-mediated proliferative block (unpublished data). Adult parasites are ideally placed within the afferent lymphatics to "bathe" the cells within the lymph nodes with down-regulatory ES. Generation of profound localized proliferative suppression by this mechanism could explain the absence of local inflammation at the site of infection in human filariasis (40, 41, 42) and in animal models (43, 44). The profundity of the suppressive mechanism(s) at play in this system is demonstrated by the fact that thus far we have been able to block proliferation of fully differentiated T cell clones, hybridomas, and primary cell lines.
L3 stage parasites must evade immune defenses on penetration of the host, on migration to the lymphatic vessels, and during growth and development toward adulthood. The observation that the infective larval stage can induce pronounced suppression of lymphocyte proliferation is intriguing given the relatively short life span of this developmental stage and its initial migratory nature (45). In particular, it indicates that cellular suppression is initiated extremely early in the course of this long term infection. In other animal models, filarial modulation of proliferative responses is generally not observed until after patency (34, 46) and thus is frequently attributed to Mf release. The results presented here suggest that immune down-modulation is a characteristic of all mammalian stages, even if it is not apparent at a systemic level until later in infection. This is consistent with work involving Brugia spp. in gerbils that has failed to show a definitive relationship between Mf and lymphocyte suppression (47, 48). Furthermore, within the peritoneal environment, Mf do not induce the profound suppression seen with adult and L3 stages but may have other as yet undefined roles in the induction of Ag-specific suppression.
The PEC suppression observed in this system is in striking contrast to
any currently defined model. For example, a suppressor cell population
is generated by infection of mice with African trypanosomes in which
nitric oxide (NO) and prostaglandins are the mediators (20). Dependence
of inducible NO synthesis on IFN- production (49) and evidence for a
direct involvement of IFN- in NO-mediated suppression of
Trypanosoma brucei infection (50, 51) suggests that it is
IFN-, not IL-4, that is necessary for induction of proliferative
block by trypanosomes. We show here and have previously shown that
neither nitric oxide, nor IFN-, nor prostaglandins are mediating
proliferative suppression by filaria-exposed PEC in vitro and that
IFN- production is also not essential in vivo (11). Similarly, the
evident importance of IL-4 in our model differs from IL-4-independent
down-regulation of proliferation in Toxoplasma gondii (52),
a process that is at least partly attributable to IFN- (53) and
IL-10 (54).
We have yet to determine the identity of the suppressive cell type but have demonstrated that the adherent PEC responsible for ablating proliferative responses in vitro is not an F4/80-positive macrophage, a further distinction from models of suppression that are dependent on suppressor macrophages for proliferative block (20, 49, 55). Additionally, the dramatic increase in eosinophils recruited to the peritoneal cavity following parasite implant raises the interesting suggestion that eosinophils are the suppressive cell type. However, the possibility remains that a minor as yet unidentified cell population is responsible for the proliferative suppression seen in this system. We are now actively investigating these possibilities.
We have established that IL-4 is critical in vivo for the induction of suppressor cells but plays no direct effector role in vitro. Similar patterns of cell recruitment can be seen in adult- and L3-implanted mice, even at low L3 numbers. One possibility is that IL-4 production early in infection may be necessary for recruitment of the appropriate cell type into the peritoneal cavity, perhaps via the production of a secondary cytokine or chemokine such as IL-5 or eotaxin. However, recruitment alone is unlikely to account for such profound suppression. The virtual absence of mast cells in implanted mice may represent mast cell degranulation, which could lead to eosinophil recruitment resulting in enhanced production of IL-4, as has been described in schistosomiasis (24). Thus, IL-4- or IL-4-driven cytokines may act alone or in concert with parasite-derived factors to directly activate recruited or resident cells to suppressor function.
The mechanism(s) of suppression exhibited by filarial-induced suppressor cells from filarial-infected mice appear to be unlike those previously described in other infectious disease systems. Further investigation of this model, as well as enhancing our understanding of filariasis, should help us to clarify the relationship of IL-4 induction to the generation of immunosuppression and may provide insight into potentially novel areas of immune modulation.
Acknowledgments
We thank Bill Gregory and Janice Murray for their maintenance of the parasite life cycle and for provision of infective larvae, and Kay Samuel and Andrew Sanderson for FACS advice. We also thank Xingxing Zang for critical comments on the manuscript.
Footnotes
1 This work was supported by the Wellcome Trust and the MRC. J.E.A. is a MRC Senior Fellow. 
2 Address correspondence and reprint requests to Dr. Judith E. Allen, Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh, EH9 3JT, Scotland, U.K. E-mail address:
3 Abbreviations used in this paper: PEC, peritoneal exudate cells; ES, excretory/secretory; D-NMMA, NG-monomethyl-D-arginine; L-NMMA, NG-monomethyl-L-arginine; L3, infective larvae; Mf, microfilariae; BmA, adult Brugia malayi Ag.
Received for publication July 7, 1997. Accepted for publication October 15, 1997.
References
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P. Loke, I. Gallagher, M. G. Nair, X. Zang, F. Brombacher, M. Mohrs, J. P. Allison, and J. E. Allen Alternative Activation Is an Innate Response to Injury That Requires CD4+ T Cells to be Sustained during Chronic Infection J. Immunol., September 15, 2007; 179(6): 3926 - 3936. [Abstract] [Full Text] [PDF]
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 C. M. Trujillo-Vargas, M. Werner-Klein, G. Wohlleben, T. Polte, G. Hansen, S. Ehlers, and K. J. Erb Helminth-derived Products Inhibit the Development of Allergic Responses in Mice Am. J. Respir. Crit. Care Med., February 15, 2007; 175(4): 336 - 344. [Abstract] [Full Text] [PDF]
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M. D. Taylor, A. Harris, M. G. Nair, R. M. Maizels, and J. E. Allen F4/80+ Alternatively Activated Macrophages Control CD4+ T Cell Hyporesponsiveness at Sites Peripheral to Filarial Infection. J. Immunol., June 1, 2006; 176(11): 6918 - 6927. [Abstract] [Full Text] [PDF]
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 M. G. Nair, I. J. Gallagher, M. D. Taylor, P. Loke, P. S. Coulson, R. A. Wilson, R. M. Maizels, and J. E. Allen Chitinase and Fizz Family Members Are a Generalized Feature of Nematode Infection with Selective Upregulation of Ym1 and Fizz1 by Antigen-Presenting Cells Infect. Immun., January 1, 2005; 73(1): 385 - 394. [Abstract] [Full Text] [PDF]
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Z. Jaffar, K.-S. Wan, and K. Roberts A Key Role for Prostaglandin I2 in Limiting Lung Mucosal Th2, But Not Th1, Responses to Inhaled Allergen J. Immunol., November 15, 2002; 169(10): 5997 - 6004. [Abstract] [Full Text] [PDF]
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 X. Zang, P. Taylor, J. M. Wang, D. J. Meyer, A. L. Scott, M. D. Walkinshaw, and R. M. Maizels Homologues of Human Macrophage Migration Inhibitory Factor from a Parasitic Nematode. GENE CLONING, PROTEIN ACTIVITY, AND CRYSTAL STRUCTURE J. Biol. Chem., November 8, 2002; 277(46): 44261 - 44267. [Abstract] [Full Text] [PDF]
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S. R. Chirgwin, P. H. Elzer, S. U. Coleman, J. M. Nowling, S. D. Hagius, M. D. Edmonds, and T. R. Klei Infection Outcome and Cytokine Gene Expression in Brugia pahangi- Infected Gerbils (Meriones unguiculatus) Sensitized with Brucella abortus Infect. Immun., November 1, 2002; 70(11): 5938 - 5945. [Abstract] [Full Text] [PDF]
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M. Rodriguez-Sosa, A. R. Satoskar, R. Calderon, L. Gomez-Garcia, R. Saavedra, R. Bojalil, and L. I. Terrazas Chronic Helminth Infection Induces Alternatively Activated Macrophages Expressing High Levels of CCR5 with Low Interleukin-12 Production and Th2-Biasing Ability Infect. Immun., July 1, 2002; 70(7): 3656 - 3664. [Abstract] [Full Text] [PDF]
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A. S. MacDonald, M. I. Araujo, and E. J. Pearce Immunology of Parasitic Helminth Infections Infect. Immun., February 1, 2002; 70(2): 427 - 433. [Full Text] [PDF]
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L. Spencer, L. Shultz, and T. V. Rajan Interleukin-4 Receptor-Stat6 Signaling in Murine Infections with a Tissue-Dwelling Nematode Parasite Infect. Immun., December 1, 2001; 69(12): 7743 - 7752. [Abstract] [Full Text] [PDF]
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F. H. Falcone, P'n. Loke, X. Zang, A. S. MacDonald, R. M. Maizels, and J. E. Allen A Brugia malayi Homolog of Macrophage Migration Inhibitory Factor Reveals an Important Link Between Macrophages and Eosinophil Recruitment During Nematode Infection J. Immunol., November 1, 2001; 167(9): 5348 - 5354. [Abstract] [Full Text] [PDF]
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L. Volkmann, M. Saeftel, O. Bain, K. Fischer, B. Fleischer, and A. Hoerauf Interleukin-4 Is Essential for the Control of Microfilariae in Murine Infection with the Filaria Litomosoides sigmodontis Infect. Immun., May 1, 2001; 69(5): 2950 - 2956. [Abstract] [Full Text] [PDF]
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H. N. Ehigiator, A. W. Stadnyk, and T. D. G. Lee Modulation of B-Cell Proliferative Response by a Soluble Extract of Nippostrongylus brasiliensis Infect. Immun., November 1, 2000; 68(11): 6154 - 6161. [Abstract] [Full Text] [PDF]
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) or 50 µg/ml conalbumin (
)
(A), or HB32 cells with media (
)
(B), in the presence of PEC from control (time
point 0) or adult parasite-implanted CBA/Ca mice over a 3-wk time
course. Proliferation was measured by [3H]TdR
incorporation and is shown as counts per minute. Data presented are
mean ± SD of three to four individual mice separately assayed.
Ag-D10.G4 proliferation was significantly less than that of controls
after 1 wk of parasite exposure (P <
0.05).
) measured by NK bioassay
(B). PEC from implanted mice were taken 2 wk
postimplantation. These experiments were conducted in the presence of
D-NMMA (shown) or L-NMMA (not shown) with
similar results. C, Proliferation of HB32 cells in the
presence of PEC from control mice or 2-wk adult- or L3-implanted mice.
Proliferation was measured by [3H]TdR incorporation. Data
presented are mean ± SD of three to four individual mice
separately assayed. Significant proliferative suppression of the D10.G4
cell clone was achieved when cocultured with PEC from adult or 400 L3
implanted animals (P < 0.05). Hybridoma
proliferation was significantly reduced in the presence of PEC from
animals implanted with 10 L3 parasites (P <
0.02), in comparison to hybridoma proliferation in the presence of
control animal PEC.
),
eosinophils (