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Departments of
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Medicine and
Pathology, Channing Laboratory, Brigham and Women’s Hospital, Boston, MA 02115;
Division of Infectious Disease, Dana-Farber Cancer Institute, Boston, MA 02115;
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Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115; and
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Maxwell Finland Laboratory, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-10, but not for IL-4. The addition
of IL-2-specific Ab to T cell lysates taken from PS A-treated animals
abrogated the ability to transfer protection, whereas the addition of
Abs specific for IFN- and IL-10 did not affect protection. Finally,
administration of rIL-2 to animals at the time of bacterial challenge
prevented abscess formation in a dose-dependent manner. These data
demonstrate that PS A-mediated protection against abscess formation is
dependent upon a CD4+ T cell-dependent response, and that
IL-2 is essential to this immune mechanism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bacteroides fragilis is the most common anaerobic bacterium isolated from clinical cases of intraabdominal abscess formation (2, 3). The pathogenesis of B. fragilis in these infections is attributable to the presence of a distinct capsular polysaccharide complex (4, 5). This complex is composed of two ionically linked polymers, termed polysaccharide A (PS A)4 and PS B. Treating animals with the most potent of these saccharides, PS A, prevents the formation of intraabdominal abscesses subsequent to challenge with B. fragilis or different abscess-inducing bacteria (6). The ability of this saccharide to protect animals against abscess formation depends upon the presence of positively charged amino and negatively charged carboxyl groups associated with its repeating unit structure (7). The presence of this charge motif is rare for bacterial polysaccharides.
Attempts to define the immunologic events regulating this host response have suggested an important role for T cells (6, 8, 9, 10). However, little is known concerning the underlying mechanisms of T cell involvement. Previous studies done in rats have shown that administration of PS A shortly before or even after bacterial challenge protects against the abscess formation induced by a heterologous array of organisms, and that this protective activity is dependent upon T cells (6). These studies suggested that PS A elicits a rapid, broadly protective immunomodulatory response that is dependent upon T cells but that is not characteristic of an Ag-specific anamnestic cell-mediated immune response.
Bacterial polysaccharides are classically defined as T cell-independent Ags. The finding that PS A mediates a protective immune response that depends upon T cells prompted further investigation of the mechanism of protection. These studies demonstrate that a soluble factor released by CD4+ T cells confers protection against abscess formation. Cytokine analysis of T cells taken from PS A-treated animals and subsequent T cell transfer experiments indicates that the soluble factor responsible for mediating this activity is IL-2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PS A from B. fragilis NCTC 9343 was prepared as described previously (6, 11, 12). PS A was isolated by hot phenol/water extraction, gel filtration chromatography, and isoelectric focusing (11, 12). Polysaccharide was prepared in sterile, pyrogen-free saline for administration to animals.
Animal model for intraabdominal sepsis and challenge inocula
An animal model for intraabdominal sepsis was utilized for these studies (13). Briefly, male Wistar rats (180–200 g, Charles River Laboratories, Wilmington, MA) were anesthetized with a single i.p. injection of 0.15 ml of Nembutal (50 mg/ml, Abbott Laboratories, North Chicago, IL). An anterior midline incision (0.5 cm) was made through the abdominal wall and peritoneum, and a gelatin capsule with 0.5 ml of inoculum was inserted into the pelvis. The inoculum contained an axenic culture of B. fragilis NCTC 9343 (108 CFU/rat) and was mixed with sterile rat cecal contents as described previously (6, 14). Sterile cecal contents are included as an adjuvant in these studies to help promote the formation of abscesses. Implantation of B. fragilis with the adjuvant results in abscess formation in 70–95% of animals, whereas implantation of the adjuvant alone does not induce abscess formation. Incisions were closed with 3.0 silk sutures, and animals were returned to cages. Animals that did not survive this surgical procedure were removed from the experiment. At 6 days postchallenge, surviving animals were necropsied in an observer-blinded fashion and examined for the presence of intraabdominal abscesses. The presence of one or more abscesses in an animal was scored as a positive result.
T cell transfer studies
Cell transfer experiments were performed as described previously (6). Animals were treated s.c. with a total of four doses of PS A (10 µg/dose) for 1 week before the harvesting of spleens. Spleens were removed from PS A-treated or saline-treated rats, counted using a Coulter FN counter (Coulter Electronics, Hialeah, FL), and examined for viability by trypan blue exclusion. The preparation was enriched for T cells by passage over nylon wool columns (>95% pure T cells as assessed by FACS analysis). T cells were fractionated by treatment with specific Ab for CD4+ or CD8+ T cells (Biosource International, Camarillo, CA) and by negative selection with magnetic beads (Perseptive Diagnostics, Cambridge, MA) as described previously (15, 16). Confirmation of purified cell populations following magnetic bead separation was performed by FACS analysis; this confirmation showed that respective cell populations were >95% pure. Purified T cells were then counted and adjusted to appropriate cell number (3 x 106/animal) before intracardiac transfer to animals (0.2 ml). Animals were challenged with bacterial inocula 24 h later.
Lysates of T cells were generated by subjecting enriched T cell
populations to a freeze/thaw cycle three times. Cell debris was
centrifuged (3000 x g), and the remaining lysate was
used for in vivo T cell transfer studies. For Ab neutralization
studies, the equivalent of 3 x 106
cells/animal was mixed with 50 µg of the appropriate Ab for 30 min at
room temperature and administered via the intracardiac route.
Polyclonal Ab specific for IL-2 (BioSource International) and mAbs
specific for IL-10 and IFN- (PharMingen, San Diego, CA) were used
for neutralization experiments. Isotype-matched rat Abs were used as
negative controls.
Cytokine mRNA analysis of T cells
A semiquantitative RT-PCR assay was employed to determine
relative mRNA expression for IL-2, IFN-, IL-4, and IL-10 in PS
A-treated and saline-treated rats. Total cellular RNA was collected
from the purified T cells using an RNeasy Mini kit (Qiagen, Santa
Clarita, CA). Briefly, 1 x 107 cells were
lysed, homogenized by repetitive passage through a 20-gauge needle, and
applied to an RNA affinity column. Residual DNA was digested with DNase
I (Life Technologies, Rockville, MD), and the RNeasy kit was used to
purify the RNA. After RNA integrity was confirmed by electrophoresis on
a 1% (w/v) agarose gel, reverse transcription was performed using the
superscript RT-PCR kit (Life Technologies). RNA in 10-µg aliquots was
primed with oligo(dT), and reverse transcription was performed
according to the manufacturer’s instructions. The resulting cDNA was
treated with RNase (Life Technologies), and PCR was performed in a
50-µl reaction volume containing 1.5 mM MgCL2,
20 mM Tris-HCl, 0.2 mM dNTP, 0.1% Triton X-100, 2.5 U Taq
polymerase, 200 ng of cDNA, and 200 ng of each primer. Stepdown PCR,
which is a simplified version of touchdown PCR, was implemented to
reduce the formation of nonspecific products (17). A hot
start was performed at 94°C for 4 min. Cycling conditions consisted
of 1 min of denaturation at 94°C, 2 min of annealing with three
cycles at each annealing temperature (67°C, 64°C, 61°C, 58°C,
55°C, and 51°C) and 3 min of extension at 72°C. An additional 20
cycles were done with an annealing temperature of 52°C, for a total
of 38 cycles. For IL-4, PCR was performed at an annealing temperature
of 58°C for 35 cycles. These conditions were identical for mRNA
samples taken from saline- or PS A-treated animals. Intron spanning
primers were designed using the prime program included in the GCG suite
of programs: ß-Actin (sense primer, 5'-CCA ACC GTG AAA AGA TGA
CCC-3'; antisense primer, 5'-TCG TAC TCC TGC TTG CTG ATC C-3'), IL-2
(sense primer, 5'-ACG CTT GTC CTC CTT GTC AAC-3'; antisense primer,
5'-CCA TCT CCT CAG AAA TTC CAC C-3'), IL-4 (sense primer, 5'-GCT GTC
ACC CTG TTC TGC TTT C-3'; antisense primer, 5'-TCA TTA ACG GTG CAG CTT
CTC-3'), IL-10 (sense primer, 5'-ACA ATA ACT GCA CCC ACT TCC-3';
antisense primer, 5'-AAA TCA TTC TTC ACC TGC TCC-3'), IFN- (sense
primer, 5'-CCA TCA GCA ACA ACA TAA GTG TC-3'; antisense primer, 5'-ACT
CCT TTT CCG CTT CCT TAG-3'). Primers were designed according to the
known DNA sequences of IL-2, IFN-, IL-4, and IL-10. Rat T cell
stimulation assays resulting in the specific release of these cytokines
were performed as positive controls to verify primer specificity and
the predicted PCR product size. Negative controls without cDNA were
amplified for every PCR experiment. The cDNA products were visualized
by electrophoresis on 1.5% agarose gels following staining with
ethidium bromide.
Statistical evaluation
Comparison of abscess formation between groups of animals was
made by 
2 analysis as supplied on commercially
available statistical software (InStat, GraphPad Software, San
Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The phenotype of T cell that mediate protection against abscess
formation in the rat model was determined. Animals were treated with
saline or PS A, and their splenic T cells were harvested. The enriched
T cell populations were then depleted of CD4+ or
CD8+ cells, and the remaining cells were
transferred to naive recipients via the intracardiac route. FACS
analysis of depleted cell populations demonstrated that >95% of
purified CD4+ or CD8+ T
cells were transferred to animals 1 day before challenge with B.
fragilis. The number of animals with abscesses per group was
determined 6 days later. Results are shown in Table I
. Animals receiving unfractionated T
cells from saline-treated animals developed abscesses (84% abscess
rate), whereas only 28% of animals receiving unfractionated T cells
from PS A-treated animals formed abscesses (p =
0.0001). The transfer of CD4+ T cells from PS
A-treated animals reduced the rate of abscess formation in recipient
animals to 29% (p = 0.0001), whereas animals
receiving CD8+ T cells had a 75% abscess rate.
The number of animals receiving CD8+ T cells from
PS A-treated rats that developed abscesses was significantly higher
than animals receiving CD4+ T cells from
similarly treated animals (p < 0.005).
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To further characterize this protective activity, a
CD4+ T cell population taken from saline- or PS
A-treated animals was fixed with 1% paraformaldehyde or subjected to a
freeze/thaw procedure to lyse cells. The subsequent fixed cell
population or cell lysate was transferred to naive recipient animals
24 h before challenge as described above. Results are shown in
Table II. Animals given untreated, lysed,
or fixed cells from saline-treated rats developed abscesses (72%,
90%, and 75%, respectively). Transfer of intact
CD4+ T cells or lysates of
CD4+ T cells from PS A-treated rats conferred
protection in naive T cell recipients (22% and 17% abscess rate,
respectively). However, fixation of the CD4+ T
cells taken from PS A-treated animals abrogated the protective
activity, yielding an 88% abscess rate compared with 75% in animals
given fixed saline-treated CD4+ T cells.
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Animals were treated with PS A as described above for T cell
transfer experiments, and RT-PCR analyses were performed on purified
splenic T cells. Results are shown in Fig. 1. Elevated mRNA levels of the Th1
cytokines IL-2 and IFN- were detected from T cells taken from PS
A-treated animals. In addition, transcript for the Th2 cytokine IL-10
was also observed. The presence of transcript for IL-4 was not noted
from these T cell preparations. Analysis of T cells from saline-treated
animals did not demonstrate mRNA transcript for IL-2, IFN-, IL-4, or
IL-10.
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To assess the role of cytokines in the transfer of protection, T
cell lysates were treated with Abs to neutralize specific cytokines. T
cell lysates from PS A- or saline-treated animals were mixed with Abs
specific for IL-2, IFN-, or IL-10. Addition of IFN-- or
IL-10-specific Ab to T cell lysates taken from PS A-treated animals did
not neutralize the transfer of protection against abscess formation
(Table III). Mixing of these
cytokine-specific Abs with T cell lysates from saline-treated animals
did not alter the ability of recipient animals to form abscesses
postchallenge. However, mixing IL-2-specific Abs with T cell lysates
from PS A-treated animals abrogated the protective activity (Table III). Transfer of PS A lysates mixed with IL-2-specific Ab resulted in
a 76% abscess rate, compared with an 11% rate of abscess formation in
animals receiving PS A lysates mixed with an isotype-matched control Ab
(p < 0.0005).
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To demonstrate the role of IL-2 in conferring protection against
abscess formation, we performed experiments in which rIL-2 was
administered to animals via the intracardiac route at the time of i.p.
challenge with B. fragilis. Animals receiving 100 pg of IL-2
had a significantly lower rate of abscesses compared with animals
receiving saline (Table IV, expt. A, 27%
vs 70%, p < 0.005).
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, expt. B. Animals receiving 1000 or 100 pg of IL-2
had significantly fewer abscesses than those receiving saline
(p < 0.002), whereas a dose of 10 pg failed to
confer a significant level of protection. Administration of rIL-4 (1000
pg) to animals did not protect against abscess formation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have demonstrated that CD4+ T cells transfer protection against abscess formation. The finding that lysates of CD4+ T cells also transfer protective activity suggested that a soluble factor(s) produced by these cells, possibly a cytokine(s), is the critical component. That a cytokine is likely an important mediator was further supported by abrogation of the protective activity following fixation of CD4+ T cells with paraformaldehyde. This fixation procedure cross-links cell surface proteins and prevents the release of soluble factors.
Due to the lack of immunologic reagents available for rats at the time, some earlier studies employed mouse models to investigate the role of cellular immunity in abscess formation. Work by Shapiro et al. more than 10 years ago showed that Ly 1–2+ (CD8+) T cells taken from mice treated with the complex capsular polysaccharide of B. fragilis ATCC 23745 transferred protection against abscess formation by this organism (18). Using a variation of the aforementioned mouse model, more recent studies by Sawyer et al. demonstrated a role for CD4+ T cells in the regulation of intraabdominal abscess formation (9). An increase in the number of abscesses per mouse was seen following administration of CD4+ cells to animals exposed previously to viable Escherichia coli and B. fragilis and subsequently challenged with a larger inoculum of these same bacterial species. It is important to note that there are a number of fundamental differences in the experimental design employed in the present work: 1) To address the mechanism of protection, we treated animals with PS A, the most active component of the capsular polysaccharide complex of B. fragilis NCTC 9343. This Ag has been defined structurally (20) and is distinct from either of the polysaccharides comprising the capsular complex of B. fragilis ATCC 23745 used in Shapiro’s earlier studies; 2) Unlike Sawyer’s work, we have not examined the issue of bacterial preexposure in the abdomen and its effect on abscess formation.
The rat model was employed in the present studies to examine the mechanisms of PS A-mediated protection. Recently, we have shown that the in vitro responses to PS A of human and rat T cells are similar, whereas mouse T cells do not respond. Although PS A stimulates in vitro proliferation of CD4+ T cells from both humans and rats, it stimulates B cells in mice (21).5 Because the in vitro response in rat cells was similar to human cells and a greater number of immunologic reagents for rats have recently become available, we chose to use the original model for intraabdominal abscess formation in rats as developed by Onderdonk et al. to study of the mechanism of protection (14).
The indication that a soluble factor(s) released by
CD4+ T cells conferred protection led to the
determination of the cytokine profile of these cells following
treatment of animals with PS A. Analysis of the mRNA produced by these
cells revealed a distinct cytokine pattern. Transcript for IL-2,
IFN-, and IL-10 was detected, but not for IL-4. This pattern did not
follow a classic Th1 or Th2 profile, but rather seemed to represent a
combination of the two patterns. This pattern is distinct but is
characteristic of the response observed to the superantigen
staphylococcal enterotoxin B (SEB) (22). Intracellular
FACS analysis of SEB-stimulated T cells demonstrated that individual T
cells produce these cytokines in a sequential pattern. That is, these
cells made IL-2 and IFN- early in the response, but subsequently
began making IL-10 a few days later. Because IL-10 has been shown to
inhibit Th1 responses, it was hypothesized that cells stimulated with
SEB likely produced this cytokine to help down-regulate the stimulatory
response. We are currently characterizing the temporal expression of
these cytokines in response to PS A.
Further T cell transfer experiments demonstrated that neutralization of
T cell lysates from PS A-treated animals with Ab specific for IL-2
abrogated the ability of these lysates to transfer protection to naive
animals. In contrast, mixing of IFN-- or IL-10-specific Ab did not
affect the protective activity. These experiments indicated that IL-2
was the soluble factor produced by T cells that transferred protection
against abscess formation. This finding led to experiments in which
rIL-2 was administered to animals at the time of challenge with
B. fragilis. The ability of exogenous IL-2 administration to
protect against abscess formation in a dose-dependent fashion
demonstrated the importance of the cytokine in mediating this
protective host response.
The finding that CD4+ T cells down-regulate this type of host response to bacterial infection is intriguing. Recent studies have demonstrated that certain T cells of this phenotype have a role in suppressing deleterious responses in animal models of transplantation and autoimmune disease (23, 24, 25). This work suggests that certain activated CD4+ T cells can induce nonresponsiveness in naive T cells in a manner that obviates unwanted host responses to specific Ags or alloantigens (26, 27, 28). The mechanism by which tolerance is induced in these cells is unclear, but some investigators have suggested that it occurs in a manner that is mediated through control of IL-2 secretion and up-regulation of the IL-2R CD25 on T cells (27, 29). It is possible that the transfer of PS A-activated CD4+ T cells from rats confers "protection" to naive animals in a similar manner. Transfer of protection by this cell type may serve to induce a nonresponsive state in animals, rendering them incapable of forming abscesses in response to bacterial challenge. The involvement of IL-2 in governing this protective activity further supports this possibility. This hypothesis is currently under investigation.
Recently, we have shown that PS A and other zwitterionic polysaccharides and peptides stimulate human and rat T cell activation in vitro (21).5 This activity required the presence of MHC class II APCs and could be blocked with MHC class II-specific Ab. Furthermore, human T cells stimulated with PS A in vitro transferred protection against abscess formation in vivo. These results led to our present investigation of the mechanism by which PS A elicits protection in vivo. Characterization of the cytokine profile produced by T cells taken from PS A-treated rats and the finding that CD4+ T cells from rats treated with this polymer transfer protection against abscess formation allowed us to determine that IL-2 plays a pivotal role in immunity. In addition, these results provide new insight with regard to the T cell-dependent properties of this polysaccharide and the mechanism by which its confers protection in a clinically relevant animal model. Our finding that the IL-2 produced by CD4+ T cells confers protection against abscess formation explains the immunomodulatory properties associated with PS A. Further studies are underway to determine the mechanism of T cell activation by PS A and its role in regulating abscess formation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Arthur O. Tzianabos, Channing Laboratory, 181 Longwood Avenue, Boston, MA 02115. E-mail address:
3 Current address: Department of Biochemistry, Rudman Hall, 46 College Road, University of New Hampshire, Durham, NH 03824.
4 Abbreviations used in this paper: PS A, polysaccharide A; SEB, staphylococcal enterotoxin B.
5 A. O. Tzianabos et al. Submitted for publication.
Received for publication February 24, 1999. Accepted for publication May 5, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ T cells respond to mycobacterial heat-shock protein. Nature 340:309.[Medline]
, and IL-10 by individual staphylococcal enterotoxin B-activated T helper lymphocytes. Eur. J. Immunol. 28:1534.[Medline]
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