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Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PT belongs to the A-B structure class of bacterial toxins (10). Its B-subunit binds to a receptor on the cell surface, and the enzymatically active A-subunit disrupts intracellular signaling by irreversible ADP ribosylation of the Gi-subclass of G proteins (10, 11). PT has pleiotropic effects on the immune system, such as T cell mitogenesis, augmentation of cytokine and Ab production, and the promotion of delayed type hypersensitivity responses (12, 13, 14, 15).
The mechanism by which PT facilitates the induction of EAE has mostly been attributed to its breaking down the blood-brain barrier (BBB) (16, 17). This interpretation has recently come under scrutiny, and the promotion of autoimmune Th1 immunity has been alternatively postulated as the primary mode of its action (8, 9, 18, 19). While PT seemed to induce polarized Th1 immunity in the response to autoantigens, it has recently been reported to further clonal expansion of both Th1 and Th2 cells in the immune response to foreign Ags (20, 21).
To take a new look at this issue, we have taken advantage of the sensitivity of the cytokine ELISPOT assay and tested the effects of PT on clonal sizes and cytokine differentiation of neuroantigen-reactive T cells in EAE directly ex vivo at the single-cell level (22).
The data show that PT prevents the protection conferred by injection of neuroantigens in IFA and promotes the clonal expansion of both neuroantigen-specific Th1 and Th2 cells and EAE. Furthermore, the results suggested that this effect of the toxin was mediated via the activation of APCs in lymphoid tissues and the CNS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained at Case Western Reserve University (Cleveland, OH) under special pathogen-free conditions. All animal procedures were conducted according to guidelines of the Institutional Care and Use Committee of Case Western Reserve University. Female SJL/J mice were injected at 6–10 wk of age with the Ag in IFA or CFA. PT (200 ng; List Biological Laboratories, Campbell, CA) was injected i.p. in 500 µl saline at 0 and 24 h, or at the time points indicated. PLP peptide AA139–151 (PLPp139–151, HSLGKWLGHPDKF) was synthesized by Princeton Biomolecules (Langhorne, PA). MBP was prepared as described (23). IFA was purchased from Life Technologies (Grand Island, NY), and CFA was made by mixing Mycobacterium tuberculosis H37RA (Difco, Detroit, MI) at 5 mg/ml into IFA. Ags were mixed with the adjuvant to yield a 2 mg/ml emulsion, of which 50 µl was injected either i.p. or s.c. as specified.
Evaluation of clinical disease
Mice were monitored daily for 30+ days after injection of neuroantigen, and the severity of disease was recorded according to the following scale (24): grade 0, no abnormality; grade 1, limp tail; grade 2, moderate hind limb weakness; grade 3, complete hind limb paralysis; grade 4, quadriplegia or premoribund state; grade 5, death. If necessary, food was provided on the cage floor.
Cell preparations from the organs tested and T cell separation
Single-cell suspensions of cells from the spinal cord were prepared as previously described (25). Afterward, the cells were plated at various concentrations ranging from 2 x 104 to 3 x 105cells/well in HL-1 serum-free medium (BioWhittaker, Walkersville, MD) together with Ag and 5 x 105 irradiated naive spleen cells as APCs. Single-cell suspensions from the spleen were prepared as previously described (20). The cells were counted and plated with Ag at 1 x 106 cells/well. For the cell separations, CD4+ T cells were obtained by passing the spleen cells through a murine CD4+ or CD8+ T cell enrichment column (R&D Systems, Minneapolis, MN) following the manufacturer’s suggested protocol, or by FACS sorting. This protocol usually results in 90–99% enrichment for CD4+ T cells. Afterward, the cells were plated at 5 x 105 cells/well in HL-1 serum-free medium together with Ag and 3 x 105 irradiated naive spleen cells as APCs.
Adoptive transfer of PLPp139–151-specific T cells
For adoptive transfer experiments, splenic mononuclear cells
from mice immunized (as indicated in Fig. 4
) were prepared as
described above. These cells were subsequently preactivated by
incubation with PLPp139–151 at the previously established optimal
stimulatory concentration (30 µg/ml) in complete DMEM for 3 days
before i.p. injection into the recipient animals. PT (200 ng) was
injected (as detailed in Fig. 5) into the donor or recipient
mice. The recipients were monitored daily after the injection, and
clinical disease was assessed according to the standard scale outlined
above. For the experiments shown in Fig. 4, measurements for all
cytokines by ELISPOT as described below were conducted before the
incubation to assess the number of cytokine-producing T cells at the
single-cell level. Subsequently, the total number of T cells injected
was adjusted according to the number of IFN-
-producing cells
calculated from the preincubation ELISPOT assays as a function of the
total number of cells recovered.
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ELISPOT plates (ImmunoSpot; Cellular Technology, Cleveland, OH)
were coated overnight with IFN--specific (R46A2; 4 µg/ml) or
IL-5-specific (TRFK5; 5 µg/ml) capture Ab, or with a combination of
both for double-color ELISPOT assays as described (20).
The plates were blocked with 1% BSA in PBS for 1 h at room
temperature, then washed four times with PBS. Spleen cells were plated
at 106 cells/well alone or with Ag (7 µM) in
HL-1 medium and cultured for 24 h for IFN-, 48 h for IL-5,
or 36 h for double-color assays. Subsequently, the cells were
removed by washing and the biotinylated detection Ab XMG1.2-biotin (2
µg/ml) for IFN- and TRFK4-biotin (2 µg/ml) for IL-5 were added
and incubated overnight. The plate-bound second Ab was then visualized
by adding streptavidin-alkaline phosphatase (DAKO, Carpinteria, CA) and
nitroblue tetrazolium (Bio-Rad, Hercules,
CA)/5-bromo-4-chloro-3-indolyl phosphate substrate
(Sigma-Aldrich, St. Louis, MO). Image analysis of ELISPOT assays was
performed on a Series 1 ImmunoSpot Image Analyzer (Cellular
Technology). In brief, digitized images of individual wells of
the ELISPOT plates were analyzed for cytokine spots, based on the
comparison of experimental wells (containing T cells and APCs with Ag)
and control wells (T cells and APCs but no Ag; usually fewer than five
spots per well). After separation of spots that touched or partially
overlapped, nonspecific noise was gated out by applying spot size and
circularity analysis as additional criteria. Spots that fell within the
accepted criteria were highlighted and counted. Additionally, spot-size
histograms were generated, reflecting the distribution of spots
according to the cytokine output per cell. The stimulation index was
calculated by dividing the number of cytokine spots detected in
experimental wells (T cells, APCs, and relevant Ag) by the number of
cytokine spots in control wells (T cells, APCs, no Ag). The spot number
in unimmunized or control mice (immunized with irrelevant Ag) was in
the same range as in the medium controls (fewer than five spots). Where
indicated, statistical analysis was performed with the paired
t test or the Mann-Whitney U rank sum test using
SigmaStat software (SPSS, Chicago, IL).
Flow cytometry analysis
Single-cell suspensions were incubated at 1 x 106 cells per sample with 0.5–1 µg of anti-I-As, anti-B7-1, anti-B7-2, CD40, or MAC-1 Abs, (BD PharMingen, San Diego, CA) for 1 h on ice. Cells were washed with PBS. Erythrocytes were lysed with Immuno-lyse (Coulter, Miami, FL) and the cells were fixed in Coulter Clone fixative reagent and analyzed on a FACSScan (BD Biosciences, Mountain View, CA).
Histopathology
At the time of the experiment the brain and spinal cord of the mice were removed and either preserved in Z-Fix or snap-frozen in 2-methyl-butane. Thin slices of the CNS tissue were prepared and stained with H&E. The tissue was then examined by light microscopy in a blinded fashion by a neuropathologist and evaluated for the extent of inflammation and graded as follows: 0, no inflammation; 1, a few mononuclear cells; 2, organization of inflammatory infiltrates around positive vessels; 3, extensive perivascular cuffing with extension into the subarachnoid space; 4, extensive perivascular cuffing with increasing subarachnoid inflammation (24).
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Results |
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Injection of neuroantigens in IFA is an efficacious way to induce tolerance and to protect rodents from EAE (5, 6). Although the mechanisms underlying this IFA-mediated protection have remained unresolved, immunoregulation, induction of immune deviation (neuroantigen-specific Th2 immunity), and a decrease in the clonal sizes of encephalitogenic neuroantigen-specific Th1 cells have been implicated (5, 6). Thus, we used the injection of neuroantigens in IFA as an experimental system to investigate the mechanism by which PT contributes to the induction of EAE.
Shown in Fig. 1A are groups of
SJL mice injected with the immunodominant
H-2s-restricted PLPp139–151 peptide in IFA,
with or without PT, and observed for up to 6 mo for clinical
symptoms of EAE. SJL mice injected with PLPp139–151:CFA and PT (a
standard protocol for EAE induction in this mouse strain) served as the
positive control for disease in these experiments (Fig. 1A, •).
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A, 
) developed relapsing remitting EAE, with similar
onset and severity to the disease observed in
PLPp139–151:CFA:PT-injected mice (Fig. 1A, •;
p > 0.05). Confirming previous reports, injection of
SJL mice with PLPp139–151:IFA without PT did not induce EAE (Fig. 1A, 
) (5, 6). Histologic examination of
brain sections revealed extensive perivascular and periventricular
mononuclear inflammatory infiltrates in both the PLPp139–151:IFA:PT-
and the PLPp139–151:CFA:PT-injected mice, but not in mice that
received PLPp139–151:IFA in the absence of PT (data not shown).
Previously it was reported that SJL mice possess high frequencies of
PLPp139–151-specific precursor T cells (26), which could
have contributed to the effect of PT on EAE induction in this model.
Therefore, we tested SJL mice for the induction of EAE with MBP, for
which an increase in the frequencies of Ag-specific precursor T cells
was not reported. As shown in Fig. 1B, EAE could also be
induced in SJL mice immunized with MBP:IFA:PT (Fig. 1B,
). However, the onset of disease was delayed in MBP:IFA:PT-injected
mice as compared with MBP:CFA:PT-immunized animals
(p < 0.005), whereas EAE severity was similar
in both groups. In contrast, mice injected with MBP:IFA without PT
never developed any clinical signs of EAE (data not shown).
Taken together, the results showed for the first time that PT prevented IFA-mediated protection from EAE in mice, expanding observations in experimental autoimmune uveitis and EAE in rats (9, 19, 27).
PT induces clonal expansion of PLPp139–151-specific Th1 and Th2 cells
The injection of prototypic foreign and self Ags such as HEL, MBP,
or PLP in IFA elicited highly polarized Th2 responses (IL-4 and IL-5,
but not IFN- production; IgG1 and IgE, but not IgG2a Abs) (6, 28). However, when PT was coinjected, Ag-specific Th1 and Th2
immune responses to foreign Ags were induced (20, 21). In
contrast, coinjection of PT with self Ags has been reported to sway the
immune response toward a Th1 pattern of cytokine production (8, 9, 19). Thus, we tested whether the induction of EAE in our
model was associated with a Th1 polarization of the autoimmune T cell
response.
Shown in Fig. 2 are the results of SJL
mice injected with PLPp139–151 in CFA or IFA, with or without
coinjection of PT. The mice were tested 2–7 wk later for
PLPp139–151-induced cytokine production. As shown in Fig. 2A, mice injected with PLPp139–151 in IFA mounted a strong
Th2 recall response (Fig. 2A, left panel,
IL-5+IFN--), whereas
mice immunized with the peptide in CFA (prototypic Th1 adjuvant)
mounted a Th1 response (Fig. 2A, middle panel,
IFN-+IL-5-).
Interestingly, the mice immunized with PLPp139–151:IFA and coinjected
with PT demonstrated a vigorous concurrent peptide-specific Th1 and Th2
response (Fig. 2A, right panel, both IFN- and
IL-5). As shown in Fig. 2B, cell separation in conjunction
with serial dilution experiments demonstrated that the cytokine
response was mediated by individual CD4+ T cells,
consistent with previous reports (29, 30, 31). Similar results
were obtained when the mice were tested at various time points after
immunization, or when different mouse strain and Ag combinations were
tested (e.g., B10.PL or C57.BL/6 mice with MBP or myelin
oligodendrocyte glycoprotein, respectively; data not shown), indicating
that the results were not particular for the SJL/PLP model.
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PT-induced Th2 cells migrate to the CNS but fail to prevent EAE
In the presented experiments, EAE was induced in
PLPp139–151:IFA:PT-injected mice in the face of high frequencies of
neuroantigen-specific Th2 cells (Fig. 2). Therefore, the
PLPp139–151-reactive Th2 cells failed to provide protection in this
model, which could have been the result of PT-induced changes, such as
impaired production of regulatory cytokines or a failure to migrate to
the brain. To test whether the PLPp139–151-specific Th2 cells actually
migrated to the CNS and exerted effector functions, we isolated spinal
cord cells from these mice and performed ELISPOT assays for Th1 and Th2
cytokines as described previously (25). As shown in Fig. 3, both PLPp139–151-specific Th1 and Th2
cells were detected in the spinal cord isolates of
PLPp139–151:IFA:PT-primed mice, as indicated by vigorous IFN- and
IL-5 production (Fig. 3, right bars; also IL-2 and IL-4, not
shown), but not in PLPp139–151:IFA- or PLPp139–151:CFA:PT-injected
animals. However, the ratio of Th1:Th2 cells in the CNS was increased
as compared with the spleen (IFN-:IL-5 ratio of 0.6 in the spleen
and 3.4 in the CNS). To rule out that IFN- and IL-5 were actually
produced by the same T cells (Th0 cells), we performed two-color
cytokine ELISPOT assays on the spinal cord isolates (data not shown).
The results demonstrated that IFN- and IL-5 were produced by
individual Th1 or Th2 cells, but not by Th0 cells, producing both
cytokines simultaneously (data not shown).
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, left bars) or in
the CNS of PLPp:IFA:PT- or PLPp:CFA:PT-injected mice before the onset
of EAE (data not shown). Second, the frequencies of
PLPp139–151-reactive cytokine-producing cells in the blood were
similar to the frequencies noted in the spleen (data not shown).
However, the clonal sizes detected in the spinal cord isolates were 5-
to 15-fold higher than in the blood or the spleen (Fig. 2A
vs Fig. 3). Finally, the ratio of CD4+ T cells to
erythrocytes in the spinal cord isolates was 1:2, whereas in the
peripheral blood it was usually <1:3500, indicating that only
approximately one of 1000 cytokine-producing cells in the CNS was
derived from the peripheral blood (25). Hence, the Th2 cells migrated to the CNS, and they were not impaired in their ability to release Th2 cytokines. The data suggest that an increased ratio of neuroantigen-specific Th1:Th2 cells may have accounted for the inability of the Th2 cells to prevent disease in this model. Alternatively, PT could have contributed to the induction of EAE via a novel, hitherto unidentified mechanism that allowed pathogenic Th1 cells to evade the regulatory effects of Th2 cells.
The encephalitogenic potential of PLPp139–151-reactive Th1 cells induced by injection with neuroantigen in IFA and PT is similar to that of T cells induced by CFA
To test whether PT had altered the effector function of
neuroantigen-specific Th1 cells, we primed SJL mice with
PLPp139–151:IFA:PT, or with PLPp139–151:CFA:PT, and tested for EAE
induction by adoptive transfer of spleen cells to naive SJL recipients.
To directly compare the encephalitogenic potential of the T cells
generated by either immunization protocol, we measured the number of
PLPp139–151-specific cytokine-producing T cells recovered from the
donor mice. For this, the recovered cells were tested in cytokine
ELISPOT assays for PLPp139–151-specific T cell responses in parallel
to the in vitro restimulation culture of the cells with the PLP
peptide. Subsequently, we calculated the total number of activated
PLPp139–151-specific T cells in culture, as outlined in
Materials and Methods, and transferred identical numbers of
PLPp139–151-reactive IFN--producing T cells from either group to
the respective naive SJL recipients. Shown in Fig. 4 are the results for SJL mice that
received increasing numbers of cells from either PLPp139–151:IFA:PT-
or PLPp139–151:CFA:PT-primed donors. Interestingly, transfer of as few
as 500 activated PLPp139–151-specific IFN--secreting T cells from
either group was sufficient to induce EAE (Fig. 4, 
). Furthermore,
disease severity increased similarly as a function of the number of
transferred cells, and disease onset was identical. In contrast,
adoptive transfer of PLPp139–151:IFA-primed T cells (no PT coinjection
of the donor mice at the time of primary immunization) did not induce
EAE (data not shown).
Taken together, the results demonstrate that T cells from PLPp139–151:IFA:PT-primed mice were as efficient in inducing EAE as were T cells from PLPp139–151:CFA:PT-primed animals. Furthermore, the data show that once PLPp139–151-specific Th1 cells were generated, PT was no longer required for the induction of EAE in the adoptive hosts. Thus, the results suggest that PT acted in the induction of EAE in this model independently of the BBB, and that the effector function of the PT-induced pathogenic Th1 cells was not changed.
IFA-induced PLPp139–151-specific Th2 cells are not rendered pathogenic by PT
Autoantigen-reactive Th2 cells have mostly been associated with
protection from autoimmune disease (32, 33, 34). However, it
has recently been shown that Th2 cells can mediate autoimmune pathology
in some models (35, 36). Because we failed to demonstrate
protection by the PLPp139–151-specific Th2 cells in our system, we
asked whether or not they contributed to the disease. Because the
separation of PT-induced Th2 cells from the concurrently induced Th1
cells was not feasible, we alternatively generated
PLPp139–151-specific Th2 cells by priming with the peptide in IFA and
then tested the effects of PT on these cells in vivo or in vitro. To
test PT effects on Th2 cells in vivo, we injected SJL mice with the PLP
peptide in IFA or CFA and waited until PLPp139–151-specific Th2 or Th1
immunity was firmly established (after 2 wk). On days 18 and 19 after
immunization, the mice were injected with PT. As shown in Fig. 5A, injection of PT into
PLPp139–151:IFA-primed mice at this late time point did not induce EAE
(Fig. 5A, ) or Th1 immune responses (data not shown). In
contrast, mice injected with PLPp139–151 in CFA developed EAE, which
was not significantly altered by the coinjection of PT (Fig. 5A, 
vs •). Similar results were obtained when PT was
injected into the recipients at the time of adoptive transfer of
PLPp139–151:IFA-primed Th2 cells into naive SJL mice (Fig. 5B, ). Similarly, in vitro incubation of
PLPp139–151:IFA-induced Th2 cells with PT and adoptive transfer to
naive recipients did not induce EAE (Fig. 5C), even at
concentrations of the toxin that were mitogenic in vitro (data not
shown) (12).
Taken together, the data show that IFA-induced PLPp139–151-specific
Th2 cells were not encephalitogenic in this model, and exposure of the
Th2 cells to PT in vivo and in vitro did not render them pathogenic.
Further supporting this conclusion was the fact that the severity of
EAE in the presented experiments was absolutely dependent on the
frequency of transferred PLPp139–151-specific Th1 cells (Fig. 4),
irrespective of the presence of Th2 cells in PLPp139–151:IFA:PT-primed
donors.
PT activates APCs in the spleen and CNS
The presented experiments showed that PT induced Th1 differentiation and clonal expansion of both self (neuroantigen)-specific Th1 and Th2 cells, similar to its effects in the T cell response to prototypic foreign Ags (20, 21). It was suggested that the underlying mechanism involved G protein-mediated activation of APCs (20). Hence, we tested whether PT acted similarly on APCs in the autoimmune response to neuroantigens, which could be a mechanism contributing to its efficacy in the induction of EAE.
Shown in Fig. 6 is the expression of
surface molecules on spleen and spinal cord cells of SJL mice after a
24-h incubation with medium (Fig. 6, upper rows) or PT (Fig. 6, lower rows, 400 ng/ml). The results show that PT enhanced
the expression of MHC class II, B7-1, and B7-2 molecules on spleen
cells 4- to 6-fold over medium control (Fig. 6A). Moreover,
it enhanced the expression of MHC class II, CD40, and MAC-1 molecules
on spinal cord cells (Fig. 6B). Similar results were
obtained with microglia and astrocyte cell lines (data not shown), and
we have also noted the enhanced production of IL-12 and IL-6 by these
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To induce Th1 immunity and EAE, the delivery of PT was required early
after injection of the myelin Ags in IFA; administration of the toxin
several weeks after immunization had no effect. Furthermore, PT was
injected i.p., and thus was remote from the s.c. site of Ag deposition.
Hence, this microbial product acted systemically in the induction of
EAE. This effect of the toxin has been interpreted in the past as a
result of its action on the BBB. The model held that PT opened up the
BBB by histamine-induced vascular leakage (17) and thereby
facilitated the transmigration of T cells and the induction of disease
(7, 37). However, this interpretation has been challenged
(18, 38), and our data support an additional mechanism of
its action as well. The data show that the induction of EAE was
critically associated with the Th1 differentiation and clonal expansion
of neuroantigen-specific T cells. Once a neuroantigen-specific Th1
response was established, PT was no longer required for the propagation
of disease, as demonstrated by the adoptive transfer of EAE with spleen
cells from PLPp139–151:IFA:PT-injected mice. These results are
consistent with studies in the rat, where PT induced Th1 immunity and
EAE after immunization with MBP in IFA, and with studies in
experimental allergic orchitis and experimental autoimmune
uveitis that demonstrated an enhancing effect of PT on autoimmune
disease and Th1 responses (8, 19, 27, 39). Furthermore,
mapping of EAE-modifying loci in mice has identified eae9 as
a PT-controlled locus that overrides genetic checkpoints in the
pathogenesis of EAE and is independent of the proposed PT-mediated
changes in vascular permeability (40). Interestingly,
eae9 is located in a region encoding CXCR5 and IL-18
(IFN--inducing factor).
Our data show that as few as 6000 IFN--producing
PLPp139–151-specific effector T cells induce lethal EAE, and many
fewer activated cells were sufficient to induce clinically significant
disease. Thus, the number of autoreactive T cells required to induce
autoimmune disease may be much lower than generally assumed.
However, in contrast to the above-mentioned studies, we have additionally observed a vigorous clonal expansion of myelin-specific Th2 cells, which led us to investigate the role of these cells.
Th2 cells have been associated with protection from autoimmune disease
(32, 33, 41, 42). However, this notion has come under
scrutiny. In particular, marmoset monkeys immunized with myelin
oligodendrocyte glycoprotein developed EAE that appeared to be Th2
mediated (35, 43). Furthermore, unopposed Th2
immunity, such as adoptive transfer of Th2 cells into immunocompromised
hosts (36) or induction of Th2 responses in IFN-
knockout mice (44), furthered autoimmune pathology.
Therefore, the PT-induced Th2 cells in our experiments could have
ameliorated the disease, could have had no effect, or could even have
contributed to the autoimmune pathology.
Several lines of evidence argue against a protective effect of the
PT-induced Th2 cells. First, disease severity and kinetics were similar
in Th1 polarized PLPp139–151:CFA:PT-injected and
PLPp139–151:IFA:PT-primed mice. The latter showed concomitant high
frequencies of Th1 and Th2 cells. Interestingly, both
neuroantigen-specific Th1 and Th2 cells were detected in the CNS of
PLPp139–151:IFA:PT-injected mice (Fig. 3), indicating that Th2 cells
indeed migrated to the CNS and released Th2 cytokines but did not
prevent or ameliorate the disease. Adoptive transfer of spleen cells
from both groups induced disease with similar severity and kinetics in
the recipients, despite the apparent cotransfer of Th2 cells with the
cells from PLPp139–151:IFA:PT-injected mice. These latter observations
are consistent with earlier reports that have failed to detect a
protective effect of Th2 cells when cotransferred with Th1 cells, such
as in insulin dependent diabetes mellitus models and EAE (45, 46).
Along with a lack of protection by the PT-induced Th2 cells, we found
no evidence for their contribution to the CNS pathology. Directly
testing PT-induced Th2 cells was not feasible; however, using
IFA-induced Th2 cells as a model, we noticed no effect of the toxin on
the promotion of EAE by Th2 cells (Fig. 5).
Hence, how could the lack of protection by Th2 cells in this model be
explained? First, PT could have prevented the migration of Th2 cells to
the CNS, e.g., by blocking chemokine receptors (47).
However, the presence of high frequencies of neuroantigen-reactive Th2
cells in the CNS in PLPp139–151:IFA:PT-primed mice argued against this
hypothesis, and MBP-reactive Th2 cells can migrate to the CNS
(48). Second, Th2 cells migrated to the CNS but arrived
too late to prevent Th1-mediated immunopathology via negative feedback
regulation of APCs by secretion of IL-4 and IL-10. Finally, the induced
Th2 cells may not be efficiently activated under the Ag-presenting
conditions in the CNS, and/or these cells could have released smaller
amounts of regulatory cytokines. Although we did not detect differences
in IL-5 or IL-4 production in IFA:PT- or IFA-induced T cells, the data
may not fully reflect the conditions in situ in the CNS. Furthermore,
there could be differences in the release of the regulatory cytokines
IL-10 and TGF-
, which we have not tested (49, 50, 51).
Alternatively, PT could have rendered the neuroantigen-specific Th1 cells resistant to regulatory mechanisms, such as the action of Th2 cytokines or Fas-Fas ligand-mediated apoptosis in the CNS. Consistent with this hypothesis, PT has been reported to interfere with induction of apoptosis in T cells (52, 53).
Thus, the effects of PT on the induction of EAE could be the result of direct action of the toxin on T cells or, indirectly, its effects on the CNS, or both.
PT-sensitive G proteins are expressed by a variety of cells in the CNS, including macrophages, microglia, astrocytes, and oligodendrocytes. Furthermore, previous reports indicated that the toxin exerted some of its immunological effects via activation of APCs in lymphoid tissues (20). Hence, it is possible that PT-activated APCs in the CNS contribute to the T cell-mediated pathology. Consistent with this hypothesis, we observed an increased expression of MHC class II and costimulatory molecules on PT-stimulated cell isolates from the spinal cords of SJL mice. Similar, albeit not as pronounced, effects were noted on PT-treated microglia and astrocyte cell lines (C. L. Shive and T. G. Forsthuber, unpublished observations). The concentrations of PT used in the in vitro studies were most likely higher than what APCs would be exposed to in mice in vivo. However, the toxin could be more efficacious in vivo because of additional effects of the histamine pathway, which could indirectly contribute to APC activation (54).
Thus, the data suggest the Th1 differentiation and clonal expansion of encephalitogenic T cells via PT effects on APCs as the mechanism for the efficacy of the toxin in EAE. The toxin could furthermore contribute to this process by promoting Ag recognition by pathogenic T cells on PT-activated microglia in the CNS. Finally, there could be additional effects of the toxin on the BBB, which could contribute to the disease process.
Importantly, the results show that EAE can be induced in this model in the complete absence of CFA. CFA injections generally result in severe skin pathology, including granuloma formation and necrosis, which are absent following IFA:PT injection (data not shown). Thus, induction of EAE with neuroantigens in IFA and PT could provide a useful alternative to the standard CFA-based EAE protocols.
Taken together, the data suggest an alternative mechanism by which microbial products, such as PT, could contribute to the initiation of human autoimmune disease in the absence of molecular mimicry. In this model, the priming or reactivation of autoreactive T cells may be facilitated by structurally unrelated microorganisms that have in common the activation of APCs. Therefore, therapies aimed at deactivation of APCs in target organs of the autoimmune attack could provide an alternative approach for the treatment of autoimmune disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas G. Forsthuber, Institute of Pathology, School of Medicine, Case Western Reserve University, Biomedical Research Building 936, 2109 Adelbert Road Cleveland, OH 44106-4943. E-mail address: TGF2{at}po.cwru.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; PT, pertussis toxin; BBB, blood-brain barrier; MBP, myelin basic protein; PLP, proteolipid protein.
Received for publication October 18, 2001. Accepted for publication April 30, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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usage. J. Immunol. 148:3776.[Abstract]
from an altered peptide ligand-specific Th2 cell clone down-regulate adoptive transfer of experimental autoimmune encephalomyelitis. J. Immunol. 164:3563.-deficient mice develop experimental autoimmune uveitis in the context of a deviant effector response. J. Immunol. 158:5997.[Abstract]
after antigen-specific triggering. Proc. Natl. Acad. Sci. USA 89:421., interleukin 4, and prostaglandin E expression in the brain. J. Exp. Med. 176:1355.This article has been cited by other articles:
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  K. M. Spach, R. Noubade, B. McElvany, W. F. Hickey, E. P. Blankenhorn, and C. Teuscher A Single Nucleotide Polymorphism in Tyk2 Controls Susceptibility to Experimental Allergic Encephalomyelitis J. Immunol., June 15, 2009; 182(12): 7776 - 7783. [Abstract] [Full Text] [PDF]
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 E. Adeeku, P. Gudapati, Y. Mendez-Fernandez, L. Van Kaer, and M. Boothby Flexibility accompanies commitment of memory CD4 lymphocytes derived from IL-4 locus-activated precursors PNAS, July 8, 2008; 105(27): 9307 - 9312. [Abstract] [Full Text] [PDF]
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X. Chen, O. M. Z. Howard, and J. J. Oppenheim Pertussis Toxin by Inducing IL-6 Promotes the Generation of IL-17-Producing CD4 Cells J. Immunol., May 15, 2007; 178(10): 6123 - 6129. [Abstract] [Full Text] [PDF]
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B. Amend, H. Doster, C. Lange, E. Dubois, H. Kalbacher, A. Melms, and F. Bischof Induction of Autoimmunity by Expansion of Autoreactive CD4+CD62Llow Cells In Vivo J. Immunol., October 1, 2006; 177(7): 4384 - 4390. [Abstract] [Full Text] [PDF]
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C. Cassan, E. Piaggio, J. P. Zappulla, L. T. Mars, N. Couturier, F. Bucciarelli, S. Desbois, J. Bauer, D. Gonzalez-Dunia, and R. S. Liblau Pertussis Toxin Reduces the Number of Splenic Foxp3+ Regulatory T Cells J. Immunol., August 1, 2006; 177(3): 1552 - 1560. [Abstract] [Full Text] [PDF]
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S. M. Kerfoot, M. U. Norman, B. M. Lapointe, C. S. Bonder, L. Zbytnuik, and P. Kubes Reevaluation of P-Selectin and {alpha}4 Integrin as Targets for the Treatment of Experimental Autoimmune Encephalomyelitis J. Immunol., May 15, 2006; 176(10): 6225 - 6234. [Abstract] [Full Text] [PDF]
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 N. Wettschureck and S. Offermanns Mammalian G Proteins and Their Cell Type Specific Functions Physiol Rev, October 1, 2005; 85(4): 1159 - 1204. [Abstract] [Full Text] [PDF]
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A. Nogai, V. Siffrin, K. Bonhagen, C. F. Pfueller, T. Hohnstein, R. Volkmer-Engert, W. Bruck, C. Stadelmann, and T. Kamradt Lipopolysaccharide Injection Induces Relapses of Experimental Autoimmune Encephalomyelitis in Nontransgenic Mice via Bystander Activation of Autoreactive CD4+ Cells J. Immunol., July 15, 2005; 175(2): 959 - 966. [Abstract] [Full Text] [PDF]
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 Y. Li, J. S. Heuser, S. D. Kosanke, M. Hemric, and M. W. Cunningham Protection against Experimental Autoimmune Myocarditis Is Mediated by Interleukin-10-Producing T Cells that Are Controlled by Dendritic Cells Am. J. Pathol., July 1, 2005; 167(1): 5 - 15. [Abstract] [Full Text] [PDF]
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H. H. Hofstetter, O. S. Targoni, A. Y. Karulin, T. G. Forsthuber, M. Tary-Lehmann, and P. V. Lehmann Does the Frequency and Avidity Spectrum of the Neuroantigen-Specific T Cells in the Blood Mirror the Autoimmune Process in the Central Nervous System of Mice Undergoing Experimental Allergic Encephalomyelitis? J. Immunol., April 15, 2005; 174(8): 4598 - 4605. [Abstract] [Full Text] [PDF]
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S. M. Kerfoot, E. M. Long, M. J. Hickey, G. Andonegui, B. M. Lapointe, R. C. O. Zanardo, C. Bonder, W. G. James, S. M. Robbins, and P. Kubes TLR4 Contributes to Disease-Inducing Mechanisms Resulting in Central Nervous System Autoimmune Disease J. Immunol., December 1, 2004; 173(11): 7070 - 7077. [Abstract] [Full Text] [PDF]
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A. C. Anderson, J. Reddy, R. Nazareno, R. A. Sobel, L. B. Nicholson, and V. K. Kuchroo IL-10 Plays an Important Role in the Homeostatic Regulation of the Autoreactive Repertoire in Naive Mice J. Immunol., July 15, 2004; 173(2): 828 - 834. [Abstract] [Full Text] [PDF]
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K. Darabi, A. Y. Karulin, B. O. Boehm, H. H. Hofstetter, Z. Fabry, J. C. LaManna, J. C. Chavez, M. Tary-Lehmann, and P. V. Lehmann The Third Signal in T Cell-Mediated Autoimmune Disease? J. Immunol., July 1, 2004; 173(1): 92 - 99. [Abstract] [Full Text] [PDF]
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C. Teuscher, M. E. Poynter, H. Offner, A. Zamora, T. Watanabe, P. D. Fillmore, J. F. Zachary, and E. P. Blankenhorn Attenuation of Th1 Effector Cell Responses and Susceptibility to Experimental Allergic Encephalomyelitis in Histamine H2 Receptor Knockout Mice Is Due to Dysregulation of Cytokine Production by Antigen-Presenting Cells Am. J. Pathol., March 1, 2004; 164(3): 883 - 892. [Abstract] [Full Text] [PDF]
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S. B. Su, P. B. Silver, P. Wang, C.-C. Chan, and R. R. Caspi Dissociating the Enhancing and Inhibitory Effects of Pertussis Toxin on Autoimmune Disease J. Immunol., September 1, 2003; 171(5): 2314 - 2319. [Abstract] [Full Text] [PDF]
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 J. F. Gao, S. B. Call, P. D. Fillmore, T. Watanabe, N. D. Meeker, and C. Teuscher Analysis of the Role of Bphs/Hrh1 in the Genetic Control of Responsiveness to Pertussis Toxin Infect. Immun., March 1, 2003; 71(3): 1281 - 1287. [Abstract] [Full Text] [PDF]
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W. Hou, Y. Wu, S. Sun, M. Shi, Y. Sun, C. Yang, G. Pei, Y. Gu, C. Zhong, and B. Sun Pertussis Toxin Enhances Th1 Responses by Stimulation of Dendritic Cells J. Immunol., February 15, 2003; 170(4): 1728 - 1736. [Abstract] [Full Text] [PDF]
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R.-N. E. Dogan, C. Vasu, M. J. Holterman, and B. S. Prabhakar Absence of IL-4, and Not Suppression of the Th2 Response, Prevents Development of Experimental Autoimmune Graves' Disease J. Immunol., February 15, 2003; 170(4): 2195 - 2204. [Abstract] [Full Text] [PDF]
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) or CFA:PT (•) (n = 6).
