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Allergy and Immunology Division, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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), and apoptosis. Lymphocytes in the G2/M phase of
the cell cycle and high levels of lymphocyte intracellular IL-2 were
found in the spleen but not in the granuloma. Cell trafficking
experiments showed Ag-specific recruitment of schistosomal egg Ag
(SEA)-reactive lymphoblasts into granulomas in vivo, as well as
recruitment to, residence within, and egress from granulomas in vitro.
Granuloma-derived lymphocytes were more highly activated than splenic
lymphocytes based on higher levels of CD69 and IL-2R expression. While
the granuloma microenvironment was rich in Th2 cytokines, during peak
granuloma formation, the lymphocytes per se from the spleen and
granuloma did not exhibit a dominant Th1 or Th2 cytokine profile,
producing low but similar levels of IL-4 and IFN-. The discrepancy
between high IL-2R expression and low levels of IL-2 protein production
by granuloma lymphocytes was associated with increased apoptosis in the
granuloma compared with the spleen. These findings support the
hypothesis that granulomas may play a role in the regulation of
systemic pathology in schistosomiasis by adversely affecting the
survival of SEA-reactive, immunopathogenic T
lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The T cell responses that characterize granuloma formation are very complex. The description of two subpopulations of CD4+ T cells has augmented the analysis of the cellular regulation of granuloma formation (6, 7, 8, 9, 10). Many investigators have studied cytokine production patterns in schistosomiasis and have demonstrated a shift from Th1 to Th2 cytokine profiles concomitant with egg deposition (11, 12, 13, 14). However, the majority of studies have relied on cell culture, including in vitro stimulation, or RNA hybridization techniques to assess cytokine production. Therefore, these studies could not precisely characterize the cells which were actually producing cytokines in vivo.
In this study, we examined the migration of lymphocytes to the granuloma, cell cycle kinetics, activation profiles, production of specific cytokines by lymphocytes in the spleen and granuloma, and apoptosis of lymphocytes from the spleen and granuloma using flow cytometry. We found that lymphocytes underwent IL-2-associated division in the spleen but not in the granuloma. Granuloma lymphocytes, when compared with splenic lymphocytes, were more highly activated than their splenic counterparts, but produced lower levels of cytokines and were more likely to undergo apoptosis. In vitro studies showed that, based on specific cytokine production, lymphocytes of the spleen and granuloma do not appear to be the primary cells responsible for the reported Th2 cytokine dominance in murine schistosomiasis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (Harlan Sprague-Dawley, Indianapolis, IN) were used in all experiments. Mice (5–6 wk of age) were infected with 30 cercariae percutaneously by tail immersion, according to the method of Bruce and Radke (15). Infected snails were obtained from Dr. Fred Lewis (Biomedical Research Institute, Rockville, MD).
Cell preparation
Mice were sacrificed at various times p.i., and their spleens and livers were aseptically removed. Spleens were pressed through 60 mesh stainless steel screens and the resultant cell suspension washed two times and suspended in complete medium (RPMI 1640 supplemented with 1 mM sodium pyruvate, 100 µg/ml streptomycin sulfate, 100 U/ml penicillin, 50 µg/ml gentamicin, 2 mM L-glutamine, 5 x 10-5 M 2-ME, 1% nonessential amino acids, and 10% heat-inactivated FCS). Livers were minced in a Waring blender at low speed for 15 s, and granulomas were collected by sedimentation. After extensive washing, granulomas were suspended in 10 ml complete medium and collagenase type IV (2.5 mg/ml; Sigma, St. Louis, MO), was added to both granulomas and splenic cell suspensions with subsequent incubation for 1 h at 37°C with frequent, gentle agitation. The cell suspensions were filtered through a 100-µm Nytex membrane (LabCor Products, Gaithersburg, MD) and washed in complete medium. Following RBC lysis with Tris-NH4Cl, the cells were washed and viable cells were enumerated by trypan blue exclusion.
Cell migration studies
In vivo studies: Splenocytes from S. mansoni infected and keyhole limpet hemocyanin (KLH)-immunized mice. Donor lymphoblasts were prepared from bulk cultures of splenic suspensions using equilibrium density sedimentation (16). Lymphoblast separation was performed by resuspending 15 x 106 cells/ml in a solution of BSA (density, 1.082) that was overlaid with a less dense solution of BSA (density, 1.060). Gradients were spun to equilibrium at 10,000 x g for 30 min at 4°C. The less dense lymphoblasts were removed from the interface. B cells were depleted by a selection step with CD45R (B220) micro beads (Miltenyi Biotec, Auburn, CA). The purity and viability of the lymphoblasts were determined by size, morphology, phenotypic characteristics, surface Ig staining, trypan blue exclusion, and [3H]thymidine incorporation in response to specific Ag. Using these criteria, the transferred populations were consistently >90% viable lymphoblasts.
The lymphoblasts (2 x 107) were labeled with the red fluorescent vital dye PKH-26 (2.2 x 10-6 M) (17) (Zyn-R, Zynaxis Cell Science, Malvern, PA). The labeling reaction was terminated by the addition of FCS, and unbound dye was removed by density centrifugation.
After washing, the cells were injected i.v. into the tails of recipient mice that had been exposed to 25 S. mansoni cercariae 10 wk before study or immunized with 100 µg of KLH in CFA 4 wk before study. Four days before transfer, 105 KLH-coated latex beads were injected into the portal veins of the KLH-sensitized mice. Eighteen hours after cell transfer, the animals were sacrificed and their lymph nodes, spleens, livers, and granulomas were obtained. Cell suspensions were prepared from each organ, and the number of labeled cells was determined by FACS.
In vitro studies. In vitro granulomas were studied using SEA-coated latex beads. Spleen cells from animals, previously exposed to S. mansoni for 12 wk or immunized with KLH, were added to SEA or KLH Ag-coated beads to form in vitro granulomas (18).
Small numbers (
1000) of cloned Th1 cells, reactive to either
SEA or KLH (19), were labeled with the fluorescent, hydrophobic dye,
1,1'dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI; Molecular Probes, Eugene, OR) and were added to the culture (20, 21). Individual cells were identified by fluorescence and tracked at
4-s intervals using videomicroscopy. The identity of the labeled cell
was confirmed by intermittent fluorescence microscopy. Images were
obtained with a Dage-MTI (Michigan City, IN) model 65 videocamera, a
JVC (Tokyo, Japan) Br-9000 VCR, Sony PVM-91 monitor (Tokyo,
Japan), and Opti-quip (Highland Mills, NY) infrared
stage-mounted incubator. The migratory pathway was digitized,
electronically plotted, and measured with a K&E model 620005
compensating planimeter (Keuffel and Esser, Germany). An index of
directed migration or persistence was calculated by dividing the net
migration that was directed toward the central point of the bead by the
total migratory path. An index of +1 meant that the cell migrated
directly to the bead. An index of 0 meant that the migration was
random. An index of -1 meant that the cell migrated directly away
from the bead. The migration index was calculated within concentric
rings of increasing diameter around the in vitro granuloma, i.e.,
0–25 µm, 25–50 µm, 50–100 µm, 100–150 µm,
etc.
Flow cytometry
In all flow cytometry determinations, individual isotype controls were included and photomultiplier tube voltage and gain settings initially adjusted such that isotype control values were near 10 fluorescence units. In all data reported here, lymphocyte gates were defined by forward and side light scatter characteristics, and background staining levels were between 5 and 15 fluorescence units.
Cell cycle kinetics. After fixation and permeabilization of cells (1 x 106), cellular DNA was stained by incubating cells in staining buffer (PBS + 2% FCS) containing 10 µg/ml propidium iodide (Sigma) and 5 µg/ml RNase A (Sigma) for 30 min (22) before analysis by flow cytometry. Data were analyzed using ModFit version 2.0 (Verity Software House, Topsham, ME) to determine the percentages of cells in G0/M, S or G2/M phases. Data shown are representative of at least three confirmatory experiments.
Surface staining.
Cells (106/sample) were washed once with PBS containing 2%
FCS (SB), suspended in 50 µl of SB containing 10 µg rat IgG
(Sigma), and incubated on ice for 10 min to block nonspecific binding.
Without washing, directly conjugated antisurface marker mAb (0.5
µg/106 cells) (PharMingen (San Diego, CA) 
CD3 clone
145-2C11; CD45R/B220 clone RA3-6B2; CD122 clone TM-ß1; CD69
clone H1.2F3; CD25 clone 3C7) was added and the samples were
incubated an additional 30 min on ice in the dark. The samples were
washed three times with SB and analyzed by flow cytometry. All data
were acquired and analyzed using a FACScan or FACScalibur instrument
equipped with CellQuest software, version 3.0.1 (Becton Dickinson,
Mountain View, CA).
Intracellular cytokine staining.
Cell suspensions (1 x 107/ml) were incubated in RPMI
1640 plus 10% FCS with 10 µg/ml Brefeldin A (Sigma) for 3 h at
37°C with 5% CO2. Following incubation, the cells were
harvested, washed once, and surface stained as described above. After
surface staining, cells were suspended in 1 ml of freshly prepared
2.5% paraformaldehyde (pH 7.4) and stored overnight at 4°C. The
following day, the samples were washed once and permeabilized by a
10-min incubation on ice in 1 ml of SB containing 0.1% Saponin
(Sigma). Cells were then pelleted and suspended in 50 µl SB
containing 0.1% Saponin plus 10 µg rat IgG and incubated an
additional 10 min on ice. Without washing, FITC-conjugated
anti-cytokine Abs (PharMingen IL-2 clone 54B6; IL-4 clone
11B11; IFN- clone XMG1.2) were added and samples incubated 30 min
on ice. Following two washes with SB plus 0.01% saponin, the samples
were immediately analyzed by FACS.
Apoptosis. Apoptosis was quantitated using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) methodology described by Gorczyca et al. (23), with modifications. Cells were prepared, surface stained, and permeabilized as above. Cells were then incubated with 50 µl of TdT reaction mixture containing 0.1 M sodium cacodylate (pH 7.2), 2 mM CoCl2, 0.2 mM DTT, 0.1 mg/ml BSA, 5 units TdT, and 0.3 nmol Cy5-dUTP (Amersham, Arlington Heights, IL) for 1 h at 37°C. After staining, the cells were washed three times with SB and analyzed by FACS. A control sample that contained all components of the reaction mixture except the TdT enzyme served as the negative control.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first wished to ascertain the site of generation of the
lymphocytes which were recovered from the spleens and granulomas of
S. mansoni-infected mice. Cell cycle kinetics of gated
lymphocyte populations from 8-wk-infected animals were analyzed. Spleen
and granuloma cells were sorted by flow cytometry, stained with
Biebrich scarlet and hematoxyline, and were 91–97% small lymphocytes
by morphologic criteria. As shown in Figure 1
, similar percentages of lymphocytes
were in G0/G1 phase from the spleen (44%) and
granuloma (50%). Forty percent of splenic lymphocytes (Fig. 1A) and 46% of granuloma lymphocytes (Fig. 1B)
were in S phase. In contrast a large difference was observed in the
numbers of lymphocytes in the G2/M phase of the cell cycle.
When cells, obtained from spleens and granulomas, were compared, over
16% of splenic lymphocytes were in G2/M, whereas <4% of
granuloma lymphocytes were in G2/M
(p < 0.01). These findings, coupled with the
observation that there were many more lymphocytes per 10,000 total FACS
events in spleens (3,857 ± 1,340) than in granulomas (403 ±
308) suggested that the vast majority of lymphocyte division was
occurring in the spleen while few if any lymphocytes were dividing in
the granulomas. Moreover, this division was contingent on infection by
S. mansoni since >98% of spleen cells from naive animals
were resting in the G0/G1 stage of the cell
cycle (data not shown).
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In vivo studies. Since our initial studies suggested that lymphocyte division was occurring in the spleen, we next sought direct evidence for specific migration of Ag-reactive lymphocytes from the spleen to the granulomas.
In vivo trafficking experiments were performed using SEA-reactive
lymphocytes labeled with the red fluorescent vital dye PKH-26 (17) to
determine whether these cells could be actively recruited to the
schistosome granuloma. We first confirmed that the labeling and flow
cytometry sorting procedures had no significant deleterious effects on
the function of the transferred cells. To determine whether cell
labeling altered cell function, an SEA-reactive Th1 clone, G4 (24), was
labeled with PKH-26. Viability, proliferation in response to Con A or
SEA stimulation, IL-2R expression, and in vitro granuloma formation
around SEA coated beads were examined (Table I). The viability of G4 cells was
unchanged as a result of PKH labeling. Both labeled and unlabeled G4
cells responded to Con A or SEA stimulation with similar levels of
proliferation. Similar IL-2R expression was observed in labeled and
nonlabeled cells and the in vitro granuloma index was not affected by
labeling with PKH.
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).
When anti-SEA lymphoblasts were used as the source of transferred
cells, >15% of the recovered labeled cells were found in the SEA
granulomas compared with <3% in the KLH granulomas. When anti-KLH
lymphoblasts were the source of transferred cells, 12% of the
recovered cells were located in the KLH granulomas and <3% were in
the S. mansoni granulomas. Using heat-killed anti-SEA
cells as the source of transferred cells resulted in the recovery of
<5% of the cells in the S. mansoni or KLH granulomas and
nearly 75% of the cells from the liver. Significant numbers of
heat-killed cells were also seen in histologic sections of the lung;
however, for technical reasons, these cells could not be recovered
efficiently for FACS quantitation. The distribution of SEA-reactive
lymphoblasts in normal, unsensitized animals were similar to those
observed in KLH-sensitized mice and for KLH-reactive lymphoblasts in
normal and S. mansoni-infected mice (data not shown). These
studies clearly demonstrated the preferential accumulation of
SEA-reactive lymphoblasts in S. mansoni granulomas due to
active recruitment and/retention within S. mansoni
granulomas.
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An index of directed migration was determined for cells as they
approached the in vitro granuloma. SEA-reactive T clones demonstrated
dramatically increased directed migration as they neared the source of
SEA; the SEA-coated bead (Fig. 3). The
index of directed migration approached 1.0; i.e., the SEA-reactive T
clones moved directly toward the SEA-coated bead. Conversely, T clones,
which were reactive to KLH, showed more random migratory patterns until
they came very close to the SEA-coated bead (<100 µ). In addition,
in vitro granulomas formed around KLH-coated beads, attracted cloned
anti-KLH cells from KLH-sensitized mice but did not attract the
cloned anti-SEA cells as effectively (Table II). Therefore, SEA-reactive T cells
selectively migrated toward an SEA nidus such as an in vitro granuloma.
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). The mean residence time of the SEA-reactive Th1
cells within the SEA granuloma was 38.4 ± 7.4 h. The mean
residence time of the anti-KLH cells which entered the SEA
granuloma was 6.2 ± 3.8 h. Analogous reciprocal differences
in residence times were observed for SEA-reactive cells in KLH
granulomas and for KLH-reactive cells in KLH granulomas (Table II).
Therefore, the SEA-reactive lymphocytes could be specifically
sequestered and retained in SEA granulomas (p
< 0.01).
The cells were next studied for their ability to leave the granulomas.
An index of directed migration was calculated as cells left the
granuloma. The initial egress index for SEA-reactive cloned T cells
from SEA granulomas was slow, suggesting that egress of SEA-reactive T
clones from SEA granulomas was initially retarded. Egress accelerated
as the cell moved away from the granuloma (Fig. 4) and migration was random after the
cell had moved over 300 µm from the SEA-coated bead. During the
course of the egress studies, the granulomas were actually growing in
size with the majority of cells moving toward the granulomas (18) (data
not shown). Therefore, the egress of SEA-reactive lymphocytes from SEA
granulomas was a true directed migration and not simply the result of
the granulomas disintegrating. Unlike the SEA-reactive clones,
KLH-reactive clones showed more random migratory patterns away from the
SEA-coated bead. Reciprocal egress migration patterns were observed for
SEA-reactive cells in KLH granulomas and for KLH-reactive cells in KLH
granulomas (Table II). These data indicated that the migration and
residence patterns of the SEA- and KLH-reactive clones were contingent
upon the antigenic composition of the granulomas.
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Initial studies showed that cells could be obtained from spleens injected into animals and subsequently recovered in vivo from granulomas. These trafficking patterns were confirmed by in vitro experiments. We next wished to determine whether any functional changes occurred in these lymphocytes as a consequence of their migration to and residence within granulomas. We first assessed the state of activation of lymphocytes obtained from spleens and granulomas of S. mansoni-infected mice. We used three criteria of activation: expression of the early activation marker CD 69 (25, 26); expression of a later, more functional activation marker, the IL-2R (CD25) (27, 28); and production of IL-2.
Expression of the early activation Ag CD69
Lymphocytes were assessed by multiparameter FACS for quantitative
phenotypic evidence of activation. The kinetics of surface CD69
expression by spleen and granuloma lymphocytes obtained from animals 6
to 26 wk p.i. are shown in Figure 5. Data
are expressed as the fold change in mean channel fluorescence (MCF) of
CD69-stained samples over isotype-matched control samples. A MCF of 1
indicates no significant CD69 expression. At 7 wk p.i., granuloma
lymphocytes expressed 1.5 x higher levels of CD69 than splenic
lymphocytes. CD69 expression on granuloma lymphocytes increased in
subsequent weeks p.i. and was maximal 10 wk p.i. when a 4.5-fold change
in MCF was observed. Examination of CD69 expression on splenic
lymphocytes showed a 2-fold increase in CD69 MCF very early in
granuloma formation (6–8 wk p.i.). The relatively low level of CD69
expression observed on splenic lymphocytes from 7–9 wk p.i.
corresponded with the higher levels of CD69 expression in granuloma
lymphocytes. From 8 to 10 wk p.i., CD69 expression increased on splenic
lymphocytes and reached a peak level of expression around 10.5 wk p.i.,
at the time of maximum granuloma formation. After 11 wk p.i., CD69
expression steadily declined on splenic lymphocytes whereas CD69
expression on granuloma lymphocytes remained relatively constant. Since
10,000 events were counted in each FACS determination, a 1% change in
MCF is significant. These observations suggested that lymphocytes were
more highly activated in the granuloma.
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After examination of CD69 expression, we studied the expression of
the IL-2R, a more specific marker of immune activation. Ag activation
of T lymphocytes results in synthesis of the -chain and
up-regulation of the ß- and -chains to form the high affinity,
heterotrimeric IL-2R complex (29, 30). We determined the percentage of
splenic and granuloma lymphocytes which were positive for the IL-2R
- or ß-chain as a function of weeks p.i. A single group of animals
was infected with S. mansoni and serially sacrificed. IL-2R
ß-chain (CD122) and IL-2R -chain (CD25) expression was examined on
splenic and granuloma lymphocytes (Fig. 6, A and B). The
data shown in Figure 6 are representative of at least three separate
groups of animals that were infected and serially examined at various
weeks p.i. The percentage of IL-2R ß-chain-positive lymphocytes in
the spleen remained relatively constant, around 2%, during the course
of infection. A much different result was observed in the granuloma. At
6.5 wk p.i., 4.5% of the granuloma lymphocytes were IL-2R ß-chain
positive. This percentage increased dramatically with increasing weeks
p.i., reaching 15% at 9 to 10 wk p.i. After 10 wk pi., the percentage
of granuloma lymphocytes bearing the IL-2R ß-chain began to decline
and returned to 4%. The patterns of IL-2R ß-chain expression
paralleled the kinetics of granuloma formation, which also peaked at 10
wk p.i. (31).
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-chain) was also examined at various weeks
p.i. CD25 expression on splenic lymphocytes peaked around 7 wk p.i., at
which point 25% of the lymphocytes were positive for CD25 (Fig. 6B). After 8 wk p.i., CD25 expression on splenic lymphocytes
decreased and remained relatively low during the remainder of the
infection period studied. In the granuloma at 7 wk p.i., nearly 50% of
the lymphocytes were positive for CD25, and maximal CD25 expression
occurred around 9 wk p.i. After 9 wk p.i., the percentage of
CD25+ lymphocytes decreased slightly but remained >40%,
which was 3–4 times the percentages observed in the spleen at the
same time. Table III compares the
percentages of CD25+ lymphocytes from the spleen and
granuloma at 7 to 8 wk p.i. vs 10 to 11 wk p.i. At both time points,
significantly greater percentages of granuloma lymphocytes were
CD25+ than were splenic lymphocytes. In addition, Table III
shows that while the number of CD25+ lymphocytes remains
relatively unchanged in the granuloma from 7 to 8 wk vs 10 to 11 wk, in
the spleen approximately twice as many lymphocytes were
CD25+ at 7 to 8 wk p.i. vs 10 to 11 wk p.i., suggesting
that lymphocytes may be initially activated in the spleen from 7–8 wk
p.i. and then are recruited to the granuloma. Since both the IL-2R ß-
and -chains were expressed by granuloma lymphocytes, these cells
expressed the high affinity IL-2R that characterizes activated
Ag-reactive lymphocytes.
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Our previous study using enzyme-linked immunospot (ELISPOT)
analysis showed that high levels of cytokines were produced in the
granuloma (32). Numerous other studies using ELISA assays and in situ
hybridization have ascribed similar cytokine production primarily to
Th2 lymphocytes and have suggested that cytokine production has
significant effects on both local and systemic immunity (13, 33, 34).
Therefore, we wished to determine the exact cytokine production
profiles of splenic and granuloma-derived lymphocytes in S.
mansoni-infected mice and to assess if the lymphocytes per se
expressed a Th1 to Th2 profile, as defined by IFN- or IL-4
production. We used FACS to measure intracellular cytokine production
by cells within the lymphocyte gate from acutely infected (10–12 wk
p.i.) mice. Figure 7 shows typical
cytokine staining patterns from 10 wk p.i. We measured intracellular
IL-2, IL-4, and IFN- in freshly isolated splenic and granuloma
lymphocytes without further in vitro stimulation. The results showed
relatively high levels of IL-2 in the spleen, with significantly lower
levels of IL-2 in the granuloma. Low but similar levels of IFN- were
produced by both splenic and granuloma lymphocytes. IL-4 production was
minimally elevated in granuloma lymphocytes compared with splenic
lymphocytes. However, when data were pooled from multiple experiments
(n 
5) examining lymphocytes from 10 to 12 wk p.i.,
the only statistically significant difference in cytokine production
observed was greater IL-2 production by splenic lymphocytes compared
with granuloma lymphocytes. No statistically significant differences
were measured for IL-4 or IFN-. Therefore, we observed no detectable
Th1 or Th2 cytokine production preference by the unstimulated splenic
or granuloma lymphocytes.
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If the granuloma is an immunoregulatory organelle, it may act
through the elimination of Ag-reactive lymphocytes. IL-2 withdrawal
with resultant IL-2 desaturation is known to induce apoptosis. We had
observed high levels of IL-2R expression along with low levels of IL-2
production in the granuloma lymphocytes. Therefore, we examined the
survival of lymphocytes in granulomas by measuring lymphocyte
apoptosis. Table IV shows the percentage
of apoptotic splenic and granuloma lymphocytes, the expression of
IL-2R, and the production of IL-2, measured 10 to 12 wk after exposure.
In the spleen, where IL-2 production was high and IL-2R expression was
relatively low, 6% of the lymphocytes were apoptotic
(n > 5). In the granuloma, where IL-2R expression was
high and IL-2 protein was low, creating a microenvironment relatively
deficient in IL-2, 50% of the lymphocytes were apoptotic. These
data suggested that lymphocytes in the granuloma were more likely to
undergo apoptosis than were lymphocytes in the spleen.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our initial studies were aimed at defining the site of generation of
SEA-reactive lymphocytes. While it is true that granulomas form around
eggs located in the liver, schistosome Ag and circulating immune
complexes exist in the bloodstream and Ag-reactive lymphocytes can be
found in the spleen (43, 44, 45). Therefore, it is possible that Ag-driven
cell division could occur at any site. When spleen cells from naive
animals were examined, >98% of the lymphocytes were resting in the
G0/G1 stage of the cell cycle (data not shown).
Our cell cycle kinetics data obtained from S.
mansoni-infected animals showed that 40% of splenic and
granuloma lymphocytes were in S phase but only splenic lymphocytes were
capable of reaching the G2/M phase of the cell cycle. These
results implied that lymphocytes were undergoing division in the spleen
but not in the granuloma. The absence of lymphocytes in the
G2/M phase of the cell cycle in the granuloma suggested
that the granuloma microenvironment was not conducive to successful
cell division. Cells which were in S phase in the granulomas might
either leave the granuloma to complete division elsewhere or may follow
a pathway of development which preferentially leads to their
elimination, perhaps through Ag-driven cell death (apoptosis).
If the granuloma is a regulatory organelle, it might influence pathology by locally sequestering and destroying pathogenic cells or by promoting the development of tolerogenic cells, which would leave the granuloma and act peripherally. Therefore, we examined the recruitment and migration of lymphocytes to the granuloma by means of in vivo and in vitro trafficking studies. Our trafficking data showed the preferential recruitment of SEA-reactive splenic lymphocytes to the granulomas in an antigenically specific manner. Since the circulation time in a mouse is less than 4 s, we were unable to assess directly the ability of activated lymphocytes to reenter the periphery or to undergo multiple recirculation cycles in vivo. However, our in vitro studies confirmed that cloned SEA-reactive lymphocytes migrated into granulomas, were specifically retained within granulomas, and subsequently migrated out of SEA granulomas.
Based on the expression of the activation Ags CD69 and the IL-2R, and the production of IL-2, our studies showed that lymphocytes were more highly activated in the granuloma than in the spleen. This preferential activation might be due to selective migration of the most highly activated cells from the spleen to the granulomas or due to augmented activation in the granuloma per se. Since cells demonstrating high levels of CD69 expression were never found in the spleen, the latter explanation is favored. These data further suggested that lymphocyte activation was being regulated in the granuloma. The increased levels of lymphocyte activation observed in the granuloma along with the presence of granuloma lymphocytes in the S phase but not in G2/M suggested that granuloma lymphocytes may be more susceptible to apoptosis, since prior activation and cell cycle entry are known criteria for apoptosis (46, 47, 48). In this context, the higher local Ag concentration and greater resultant T cell activation in the granulomas may be the stimulus for this apoptosis.
The host immune response to S. mansoni infection has been
shown to be a T cell-dependent process (1, 2, 24, 49). Classically, the
host initially responds with a Th1-type response which has been shown
to be directed against early stages of the parasite and to be important
for the induction of cell-mediated protective immunity to S.
mansoni (50, 51, 52). As the disease progresses, schistosomal SEA
become recognized by specific granuloma forming CD4+ T
lymphocytes (53, 54). Disease progression is also characterized by an
increasing dominance of Th2 cytokines (IL-4, IL-5,
granulocyte-macrophage CSF, IL-10) relative to Th1 (IFN-)-associated
cytokines. This transition has been attributed to a change in Th1 to
Th2 lymphocyte dominance (12, 13, 55, 56). The balance of Th1 to Th2
cytokines is important in the regulation of pathology, in particular
granuloma formation and hepatic fibrosis (12, 13, 49, 55, 57, 58, 59, 60, 61), and
may have systemic consequences as well (33, 34, 62, 63).
Other recent studies which have suggested non-CD4+ sources of IL-4 (64, 65) underscore the importance of precisely defining the cellular source(s) of cytokines in the particular experimental model being examined. In the current study we assessed lymphocyte cytokine production on a single cell basis using FACS to precisely define the cellular source(s) of the reported Th1 and Th2 cytokines at the level of 99% accuracy. We examined spleen and granuloma lymphocytes from acutely infected animals, immediately upon recovery, without further in vitro stimulation in an attempt to ascertain the true in vivo cytokine profiles as closely as possible. Our findings showed that the lymphocytes per se were not responsible for the reported Th2 cytokine dominance found in granulomas in S. mansoni-infected mice. However, we did observe a dominant Th2 cytokine profile in a nonlymphocyte population, and the results of those studies are the subject of a different report (32). The studies that have reported Th1 to Th2 cytokine dominance in lymphocytes employed artificial immunization models and/or in vitro stimulation in their cytokine assays. The significance of the potential for Th2 cytokine production after in vitro (12) stimulation and the strength of the analogy between findings based on the lung and liver granuloma models (66, 67) and in vivo events occurring as a consequence of infection, are problematic.
In our determinations, the profiles of cytokine production by splenic and granuloma lymphocytes were very similar with the exception of IL-2 production. Granuloma lymphocytes appeared to be relatively deficient in IL-2 production when compared with spleen-derived lymphocytes. IL-2 withdrawal with resultant IL-2R desaturation is known to be one mechanism whereby apoptosis is induced. In addition to IL-2 withdrawal, cellular activation and entry into the cell cycle are prerequisites for apoptosis (68, 69, 70). Since we initially observed a relative deficiency of IL-2 in the granuloma accompanied by higher levels of IL-2R expression and activation, as well as the lack of successful lymphocyte division in the granuloma, we subsequently examined lymphocyte apoptosis as a possible mechanism whereby the granuloma might regulate cell survival and subsequent immunopathology. Our initial studies clearly showed that a higher percentage of granuloma lymphocytes were apoptotic than splenic lymphocytes. We are currently investigating the possibility that cytokine production may effect subsequent apoptosis by specific subpopulations of T cells.
In summary, these data suggest that the granuloma recruits and activates lymphocytes. Moreover, the granuloma may function as an immunoregulatory organelle that regulates Ag-specific lymphocyte activation, cytokine production, and apoptosis. Splenic and granuloma lymphocytes did not exhibit a dominant Th2 cytokine production profile in these experiments, which examined cells without in vitro stimulation. We did observe a nonlymphocyte population that produced large amounts of Th2 cytokines in the granuloma, and these data will be the subject of a future report. Granuloma lymphocytes were more likely to undergo apoptosis than splenic lymphocytes, and we are investigating this phenomenon in greater detail to determine whether the granuloma might eliminate resident, potentially pathogenic, lymphocytes by IL-2-dependent, Fas/Fas ligand-mediated apoptotic mechanisms. The precise mechanism(s) of the induction of apoptosis in SEA-reactive lymphocytes sequestered in the granulomas and the systemic implications of that apoptosis are the subjects of ongoing studies.
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Footnotes |
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2 Current address: Department of Parasitology, Akita University School of Medicine, Akita, Japan.
3 Address correspondence and reprint requests to Dr. S. Michael Phillips, Allergy and Immunology Division, University of Pennsylvania School of Medicine, 518 Johnson Pavilion, Philadelphia, PA 19104. E-mail address:
4 Abbreviations used in this paper: SEA, schistosomal egg Ag; p.i., postinfection; KLH, keyhole limpet hemocyanin; DiI, 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate; SB, PBS containing 2% FCS; MCF, mean channel fluorescence.
Received for publication December 1, 1997. Accepted for publication June 9, 1998.
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(IFN-), IL-4, and IL-10 in schistosome egg granuloma formation: in vivo regulation of Th activity and inflammation. Clin. Exp. Immunol. 98:395.[Medline]
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) or
granulomas (
) at various weeks p.i. was measured by FACS. The fold
change in MCF intensity was determined by dividing the MCF of
CD69-stained cells by the MCF of an isotype-matched control tube. The
data are expressed as the fold change in that ratio. Each data point
represents the mean ± SEM of at least three observations.
) at various weeks
p.i. was measured by FACS. Data are expressed as the percentage of
lymphocytes expressing the IL-2R ß-chain or IL-2R 