Pertussis Toxin Inhibits Induction of
Tissue-Specific Autoimmune Disease by
Disrupting G Protein-Coupled Signals
This information is current as
of June 18, 2017.
Shao Bo Su, Phyllis B. Silver, Meifen Zhang, Chi-Chao
Chan and Rachel R. Caspi
J Immunol 2001; 167:250-256; ;
doi: 10.4049/jimmunol.167.1.250
http://www.jimmunol.org/content/167/1/250
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References
Pertussis Toxin Inhibits Induction of Tissue-Specific
Autoimmune Disease by Disrupting G Protein-Coupled Signals
Shao Bo Su, Phyllis B. Silver, Meifen Zhang, Chi-Chao Chan, and Rachel R. Caspi1
P
ertussis adjuvant has been used for many years to enhance
induction of organ-specific autoimmune diseases elicited
by immunization of laboratory animals with the appropriate tissue Ags. Such models include experimental autoimmune encephalomyelitis (EAE2), experimental autoimmune orchitis, experimental autoimmune uveitis (EAU), and others (1– 6). Subsequently, it
was found that pertussis toxin (PTX) is the component of Bordetella
pertussis responsible for facilitating disease development (7, 8), and
now it is used routinely for the enhancement of autoimmune diseases
in experimental animals.
PTX is a noncovalently linked heterohexameric protein that is
structurally and functionally divided into subunits A and B, similarly to other bacterial toxins such as cholera toxin (CT) and Escherichia coli heat-labile toxin (9 –11). The A subunit (A protomer)
is composed of a single peptide (S1) with ADP-ribosyltransferase
activity, that modifies GTP-binding regulatory proteins (G proteins), thus interfering with G protein-dependent signal transduction in mammalian cells. The B subunit (B oligomer), composed of
a pentameric protein complex, confers cell surface binding specificity on the toxin and delivers the A protomer into the cell (9, 11).
The mechanism by which PTX promotes the development of
autoimmune diseases in experimental animals is not fully understood. It is known that PTX has multifaceted effects that include
Laboratory of Immunology, National Eye Institute, National Institutes of Health,
Bethesda, MD 20892
Received for publication September 18, 2000. Accepted for publication April
30, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Rachel R. Caspi, Laboratory of
Immunology, National Eye Institute, National Institutes of Health, 10 Center Drive,
10/10N222, Bethesda, MD 20892-1857. E-mail address: [email protected]
2
Abbreviations used in this paper: EAE: experimental autoimmune encephalomyelitis; EAU, experimental autoimmune uveitis; PTX, pertussis toxin; IRBP, interphotoreceptor retinoid-binding protein; CT, cholera toxin; MCP, monocyte chemoattractant
protein; MIP, macrophage-inflammatory protein.
Copyright © 2001 by The American Association of Immunologists
histamine sensitization of endothelial cells, enhancement of vascular permeability, inhibition of lymphocyte recirculation, mitogenic effects on T and B cells, and other effects. Early studies by
Linthicum, Munoz, and their collaborators (12–14) uncovered effects of PTX on vascular permeability and connected susceptibility
and the effect of PTX in the EAE model to histamine sensitization
genes and the disruption of blood-brain barrier. As a result of these
studies, it has been widely accepted for over 20 years that central
to the enhancement of autoimmunity by PTX is enhancement of
vascular permeability, facilitating the breakdown of blood-tissue
barriers and promoting infiltration of inflammatory cells into the
target organ (12, 13).
The present study set out to examine directly the premise that
enhancement of vascular permeability underlies the enhancing effect of PTX on autoimmunity. We reasoned that if breakdown of
blood-tissue barriers is indeed a major mechanism, the effect of
PTX should be maximal if it is administered at the time of effector
cell migration to the target organ. Unexpectedly, administration of
PTX to mice concurrently with adoptive transfer of activated
uveitogenic T cells or 1 wk after immunization with retinal Ag in
CFA completely blocked the development of EAU. Subsequent
experiments aimed at elucidating the mechanism of this phenomenon led to the conclusion that PTX protects from EAU at least in
part by compromising migration and infiltration of effector cells to
the target organ secondary to blockade of signaling through Giprotein coupled receptors.
Materials and Methods
Animals
Six- to 8-wk-old B10.A and B10.RIII mice were supplied by The Jackson
Laboratory (Bar Harbor, ME). Animal care and use was in compliance with
institutional guidelines and with the Association for Research in Vision and
Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Ag and reagents
Peptide SGIPYIISYLHPGNTILHVD representing residues 161–180
(p161–180) of human interphotoreceptor retinoid-binding protein (IRBP)
0022-1767/01/$02.00
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Pertussis toxin (PTX) has been used for many years as an adjuvant that promotes development of tissue-specific experimental
autoimmune diseases such as experimental autoimmune encephalomyelitis, experimental autoimmune uveitis (EAU), and others.
Enhancement of vascular permeability and of Th1 responses have been implicated in this effect. Here we report a surprising
observation that, in a primed system, PTX can completely block the development of EAU. Disease was induced in B10.RIII mice
by adoptive transfer of uveitogenic T cells, or by immunization with a uveitogenic peptide. A single injection of PTX concurrently
with infusion of the uveitogenic T cells, or two injections 7 and 10 days after active immunization, completely blocked development
of EAU. EAU also was prevented by a 1-h incubation in vitro of the uveitogenic T cells with PTX before infusing them into
recipients. Uveitogenic T cells treated with PTX in vitro and lymphoid cells from mice treated with PTX in vivo failed to migrate
to chemokines in a standard chemotaxis assay. Neither the isolated B-oligomer subunit of PTX that lacks ADP ribosyltransferase
activity nor the related cholera toxin that ADP-ribosylates Gs (but not Gi) proteins blocked EAU induction or migration to
chemokines. We conclude that PTX present at the time of cell migration to the target organ prevents EAU, and propose that it
does so at least in part by disrupting signaling through Gi protein-coupled receptors. Thus, the net effect of PTX on autoimmune
disease would represent an integration of enhancing and inhibitory effects. The Journal of Immunology, 2001, 167: 250 –256.
The Journal of Immunology
was synthesized by Fmoc chemistry on a peptide synthesizer (model 461;
PE Applied Biosystems, Foster City, CA). PTX, CT, CFA, and Mycobacterium tuberculosis strain H37RA were purchased from Sigma (St. Louis,
MO). PTX B oligomer was purchased from Research Biochemicals
(Natick, MA). Murine recombinant SDF-1␣, macrophage-inflammatory
protein (MIP)-1␣, MIP-1␤, and RANTES were generously provided by
J. L. Gao and P. M. Murphy, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, (Bethesda, MD).
Uveitogenic T cell line
A long-term T cell line specific to the IRBP-derived p161–180 was established from B10.RIII mice and was propagated as described previously
(15). Briefly, a polarized Th1 cell line was established from lymph node
cells of B10.RIII mice primed with p161–180 by initially stimulating the
cells with the immunizing peptide in the presence of IL-12 and anti-IL-4
Abs. The cells subsequently were propagated by alternating cycles of stimulation with p161–180 and expansion in IL-2 containing medium every
2–3 wk. This cell line produces a typical Th1 cytokine profile and induces
EAU with a clinical onset of 4 – 6 days after transfer.
Primary culture of IRBP-specific lymph node cells
EAU induction
By adoptive transfer. Freshly stimulated uveitogenic line cells (2 ⫻ 106)
or 10 ⫻ 106 primary culture cells suspended in 1 ml of PBS with 0.5%
mouse serum were injected into the peritoneal cavity of syngeneic recipients. To test the effect of PTX on adoptive EAU, the indicated amounts of
PTX in 0.2 ml of PBS containing 1% mouse serum were infused into the
tail vein. In some experiments, freshly stimulated line cells were incubated
in vitro with the indicated amounts of PTX, CT, B oligomer, or control
medium for 1 h at 37°C before adoptive transfer.
By active immunization. EAU in B10.RIII mice was induced with 20 ␮g
of p161–180 and in B10.A mice with 50 ␮g of IRBP as a solution in PBS,
emulsified 1:1 v/v in CFA that had been supplemented with M. tuberculosis
strain H37RA to 2.5 mg/ml. A total of 200 ␮l of emulsion was injected s.c.,
divided among three sites: base of tail and both thighs. In some groups, 0.5
␮g of PTX (Sigma) was injected i.p. 7 and/or 10 days after immunization.
Eyes were collected 14 days after immunization, processed, and scored as
described below.
EAU scoring
Fundoscopic evaluation for longitudinal follow-up of disease was done
with a binocular microscope under systemic anesthesia and after pupil
dilation, as described previously (16). Scores between 0 and 4 were assigned based on the number and size of discernible lesions. Eyes for histological EAU evaluation were harvested 10 days after adoptive transfer or
14 days after active immunization. Enucleated eyes were immediately prefixed for 1 h in 4% phosphate-buffered glutaraldehyde (to prevent artifactual detachment of the retina), and then transferred to 10% phosphatebuffered formaldehyde until processing. Fixed and dehydrated tissue was
embedded in methacrylate, and 4- to 6-␮m sections were stained with
standard H&E. Eye sections cut at different planes were scored in a masked
fashion by one of us who is an ophthalmic pathologist (C.C.C.). Incidence
and severity of EAU were graded on a scale of 0 to 4 in half-point increments by the criteria described previously (16, 17), which are based on the
type, number, and size of lesions present.
Cell migration assay
Axillary and inguinal lymph node cells of mice were harvested 24 h after
i.v. administration of PTX, CT, or B oligomer. Uveitogenic T line cells
were collected 1 h after in vitro treatment with PTX. Migration of cells to
chemokines was assessed by using a 48-well microchemotaxis chamber
technique as described previously (18, 19). Briefly, a 25-␮l aliquot of chemoattractant solutions diluted in chemotaxis medium (RPMI 1640, 1%
BSA, 25 mM HEPES) was placed in the wells of the lower compartment.
A 50-␮l cell suspension (2 ⫻ 106 cells/ml in chemotaxis medium) was
placed in the wells of the upper compartment of the chamber (Neuroprobe,
Cabin John, MD). The two compartments were separated by a polycarbonate filter (5-␮m pore size; Neuroprobe) coated with 20 ␮g/ml Fibronectin (Sigma) at 4°C overnight. The chamber was incubated at 37°C for 4 h
in humidified air with 5% CO2. At the end of the incubation, the filter was
removed, fixed, and stained with Diff-Quik (Harlew, Gibbstown, NJ). The
number of migrated cells in three high-powered fields (⫻400) was counted
by light microscopy after coding the samples. Results are expressed as the
mean (⫾SD) value of the migration in triplicate samples.
Reproducibility and data presentation
Experiments were repeated at least twice, and usually three or more times.
Results were highly reproducible. Each mouse (average of both eyes) is
shown as a dot in the figures and is considered as one event for the purpose
of statistical analysis. Analysis of EAU scores was by Snedecor and Cochran’s test for linear trend in proportions (nonparametric, frequency
based; Ref. 20). Analysis of cell migration was by independent t test (twotailed). Differences of p ⱕ 0.05 were considered significant. The results of
statistical analyses are given in the figure legends.
Results
PTX administered at the time of effector cell migration aborts
induction of EAU
B10.RIII mice immunized with IRBP or with its major epitope
p161–180 are able to develop EAU without PTX treatment. However, treatment with 0.2–1 ␮g of PTX concurrently with immunization enhances disease scores (21). For EAU to develop, Agspecific effector cells primed in the periphery must migrate to the
target organ, extravasate into the tissue, and recruit inflammatory
leukocytes, a process that should be facilitated by enhancement of
vascular permeability and breakdown of the blood-organ barrier.
To evaluate the effect of PTX administered at the time of effector
cell migration on induction of EAU, B10.RIII mice immunized
with a uveitogenic dose of p161–180 were given PTX on day 7
after active immunization. This is the time considered to be critical
for effector cell migration, as clinical onset of EAU typically occurs on day 8 –9 after immunization. Eyes were collected on day
14. Unexpectedly, a single infusion of 0.5 ␮g of PTX on day 7
inhibited EAU scores in most mice, and if followed by a second
dose on day 10, largely prevented EAU in all mice (Fig. 1A). It is
important to point out that this is a dose that reproducibly enhances
EAU in a variety of mouse strains when administered on day 0,
concurrently with immunization.
In a second approach, based on adoptive transfer of Ag-specific
effector T cells, a single infusion of 1 ␮g of PTX was given to
B10.RIII mice concurrently with adoptive transfer of 2 ⫻ 106 cells
from a uveitogenic T cell line. EAU scores were assessed 10 days
later by histopathology. Again, rather than enhancing disease
scores, PTX completely inhibited expression of EAU (Fig. 1B). An
identical effect was observed with IRBP-primed lymph node cells
from B10.A mice after primary culture (data not shown).
Long-term follow-up of mice treated or not with PTX was performed by weekly fundoscopy for up to 35 days after adoptive
transfer of the T cell line. The control group developed full-blown
disease (scores of 4) within the first week (with onset occurring ⬃4
days after transfer), and remained at or close to that grade throughout the observation period. The PTX-treated group remained negative. After as long as 21 days, only about half of the mice developed minimal retinal signs initially in one eye (grade 0.5), and
subsequently their diseases stabilized with little further progress
(Fig. 2).
In a subsequent series of experiments, the effects of different
doses and timing of PTX administration on EAU induced by adoptive transfer of uveitogenic T cells were studied. Fig. 3 demonstrates that inhibition of adoptive EAU by PTX is dose-dependent
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Donor B10.A mice were immunized with 50 ␮g of IRBP. Lymph node
cells and spleen cells collected on day 14 after immunization were pooled.
The cell suspension was adjusted to 107 cells/ml in 1% normal mouse
serum-RPMI 1640 medium, and the cultures were stimulated in 75-cm2
flasks for 72 h with 30 ␮g/ml of IRBP in the presence of 50 ng/ml IL-12.
To remove excess adherent cells (macrophages), the stimulating cultures
were transferred into new flasks after 24 h and again after 48 h. After 3
days, the lymphocytes were separated from erythrocytes and debris by
discontinuous density gradient centrifugation on Ficoll (Lymphocyte M;
Accurate Chemicals, Westbury, NY) and counted in preparation for injection into recipient mice.
251
252
INHIBITION OF EAU BY PTX
FIGURE 3. Dose response of inhibitory effect of PTX on EAU.
B10.RIII mice received 2 ⫻ 106 cells from a uveitogenic T cell line i.p. and
the indicated amount of PTX i.v. at the same time. EAU score was assessed
10 days later by histopathology. The EAU scores of PTX-treated mice are
different from controls for the 1-, 0.5-, and 0.1-␮g doses (p ⬍ 0.05).
FIGURE 1. Inhibition of EAU induction by PTX. A, B10.RIII mice
were immunized with 20 ␮g/mouse of IRBP p161–180 to induce EAU.
PTX was injected by i.v. at the indicated times relative to Ag immunization. EAU score was assessed 14 days later by histopathology. The EAU
scores of PTX-treated mice at all points differ significantly from the respective controls (p ⬍ 0.05). B, B10.RIII mice received 2 ⫻ 106 cells from
a uveitogenic T cell line by i.p. injection concurrently with 1 ␮g of PTX by
i.v. EAU score was assessed 10 days later by histopathology.
and gradually diminishes over a 1-log dose difference from 1 to 0.1
␮g. Fig. 4 illustrates that the protective effect of a single 0.5-␮g
dose of PTX is detectable for almost 2 wk after its administration.
The maximal inhibitory effect was observed when PTX was given
on day 0 relative to adoptive transfer, and near maximal effect was
seen when PTX was given 3 days before adoptive transfer. Protection was still apparent with PTX given as long as 10 days before
infusion of the uveitogenic cells. Thus, the effect of PTX is
long-lived.
FIGURE 2. Long-term effects of PTX on adoptively transferred
EAU. B10.RIII mice received 2 ⫻ 106 cells from a uveitogenic T cell
line i.p. and 0.5 ␮g of PTX i.v. at the same time. Mice were followed
weekly by fundoscopic examination. The EAU scores of PTX-treated mice
vs their respective controls are significantly different at each of the time
points (p ⬍ 0.05).
The biological activities of PTX are to ADP-ribosylate Gi type G
proteins, to down-regulate cAMP production, inhibit Ca2⫹ flux,
and interfere with cell migration (9). PTX is composed of an enzymatically active subunit (A protomer) responsible for the biological activities, and a membrane binding subunit (B oligomer)
that delivers the A subunit into the cell (9, 10). The structurally and
functionally related CT ADP-ribosylates Gs type G proteins; it
increases cAMP production, and does not inhibit cell migration
and Ca2⫹ flux (9, 11). To examine whether the protective effects of
PTX involved ADP ribosylation of Gi proteins, we compared the
ability of intact PTX, of the enzymatically inactive B oligomer,
and of CT to inhibit adoptive transfer of EAU. Fig. 5A shows that
in vivo administration of the B oligomer or of CT to mice subsequently injected with uveitogenic cells did not prevent EAU, suggesting that the protective effect requires the ADP-ribosylating activity of the A subunit.
We next examined whether PTX could act directly on the
uveitogenic T cells, by preincubating line cells about to be adoptively transferred into mice with PTX in vitro for 1 h. Fig. 5B
FIGURE 4. Time dependence of protective effect of PTX. Groups of
B10.RIII mice were given 0.5 ␮g of PTX i.v. at the indicated time relative
to adoptive transfer of 2 ⫻ 106 cells from a uveitogenic T cell line i.p. EAU
score was assessed 10 days later by histopathology. The EAU scores of
PTX-treated mice are significantly different from untreated controls for all
points between 0 and ⫺10 days (p ⬍ 0.05).
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Inhibition is dependent on the enzymatic A-protomer subunit of
PTX and can be exerted directly on the T cell
The Journal of Immunology
shows that treatment of uveitogenic T cells with PTX before transfer was sufficient to inhibit adoptive EAU. Microscopic examination of the cells after the incubation for vital dye exclusion indicated that they were fully viable, making it unlikely that the
protective effect of PTX involved toxicity. This is in line with the
observations of others concerning lack of toxicity of PTX on lymphoid cells (22, 23). Again, the enzymatically inactive B oligomer
and the Gs protein-targeting CT were unable to inhibit the ability
of the T cell line to induce disease.
Uveitogenic T cells incubated with PTX as well as lymphocytes
of PTX-treated mice are unable to migrate to chemokines
Chemokines signal through Gi protein-coupled receptors, are sensitive to PTX, and play important roles in cell migration and extravasation into the tissues (24, 25). Therefore, it was logical to
suspect that PTX inhibits EAU due at least in part to effects on
chemokine signaling. To test this hypothesis, the migration of
PTX-treated T cells and of lymph node cells from PTX-treated
mice to chemokines was examined by a standard chemotaxis assay
in Boyden chambers (18). Uveitogenic T cells incubated with PTX
did not migrate to chemokines, including MIP-1␣ and RANTES
(Fig. 6A). Incubation with B oligomer or CT did not affect the
chemotactic responses of the uveitogenic line cells. Similarly,
lymph node cells from B10.RIII mice injected 24 h earlier with
PTX were compromised in their ability to respond to chemokines,
FIGURE 6. A, Incubation with PTX inhibits migration of uveitogenic T
cells to chemokines. Uveitogenic T cell line was treated with 100 ng/ml of
CT, B oligomer, or PTX at 37°C for 1 h, washed, and subjected to a
standard chemotaxis assay in a Boyden chamber toward the indicated chemokines (10 nM). B, Lymph node cells from PTX-treated mice fail to
respond to chemokines. B10.RIII mice were injected i.v. with 0.5 ␮g of
PTX, 1 ␮g of CT or B oligomer, or were left untreated. Lymph node cells
were collected after 24 h and were subjected to a standard chemotaxis
assay in a Boyden chamber. The number of migrated cells per field in
the PTX-treated group vs nontreated group is significantly different (t test,
p ⬍ 0.05).
but B oligomer and CT had no inhibitory effect (Fig. 6B). These
data support the notion that the protective effect of PTX is attributable to its enzymatic activity that disrupts Gi protein-coupled
chemokine receptor signaling.
Interference with migration of either the Ag-specific T cells or
of the recruited inflammatory leukocytes is sufficient to abort
EAU induction
Previous studies on the EAU model demonstrated that induction of
EAU after adoptive transfer of (fluorescently labeled) uveitogenic
T cells involves two discrete waves of cell entry into the eye (26).
Small numbers of labeled Ag-specific T cells infiltrate the retina
within 24 h after cell transfer and disappear by 72 h. This is followed at 96 h by a massive and prolonged influx of unlabeled
host-derived leukocytes (thought to have been recruited by the
earlier wave of Ag-specific cells) with a small proportion of labeled cells mixed in, and induction of the tissue damage typical of
EAU. We were interested in dissecting the effect of PTX on the
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FIGURE 5. A, Prevention of adoptive EAU is dependent on the A subunit of PTX. B10.RIII mice received 2 ⫻ 106 cells from a uveitogenic T
cell line i.p. The mice received 0.5 ␮g of PTX, 1 ␮g of CT, or 1 ␮g of B
oligomer i.v. at the same time. EAU score was assessed 10 days later by
histopathology. B, In vitro treatment of uveitogenic T cells with PTX before transfer is sufficient to inhibit EAU. Freshly stimulated uveitogenic T
cell line was treated with 100 ng/ml CT, B oligomer, or PTX at 37°C for
1 h and washed, and 2 ⫻ 106 cells were injected by i.p. into syngeneic
B10.RIII mice. EAU score was assessed 10 days later by histopathology.
In both panels, the EAU scores of PTX-treated groups differ significantly
from the untreated as well as the B oligomer- or CT-treated groups (p ⬍
0.05). B oligomer- and CT-treated groups do not differ from control or
from each other.
253
254
first and second stage of cell entry in terms of protection from
disease. Toward this end, mice were treated with PTX concurrently with adoptive transfer of the uveitogenic T cell line (day 0),
on day 3, or on day 3 and day 7. EAU was assessed on day 10. Fig.
7 shows that, as before, a single injection of PTX on day 0 (corresponding to the first wave of infiltration) was protective, as were
two injections on day 3 and 7 (corresponding to the second wave
of infiltration). A single injection of PTX on day 3 did not prevent
emergence of disease on day 10, consistent with the more prolonged kinetics of the second phase of infiltration. Thus, inhibition
of either the first wave (Ag-specific T cells) or the second wave
(mostly recruited host leukocytes) of cellular infiltration aborts development of EAU.
Discussion
Many rodent strains require PTX concurrently with immunization
to develop EAU, and in the ones that do not require it, PTX enhances disease scores. The enhancing effects of PTX on autoimmunity also have been well documented in EAE and autoimmune
orchitis, but the mechanism has been the subject of some controversy. Some early studies indicated effects on vascular permeability, and connected susceptibility and the effect of PTX in the EAE
model to histamine sensitization genes and the disruption of the
blood-brain barrier (12–14). Other early studies presented evidence favoring effects on the sensitization phase of immunity (27).
Our studies addressing the role of PTX in EAU were based on
two hypotheses. We reasoned that because most of the PTX injected at the time of immunization must be gone by the time that
migration of effector cells takes place into the target organ 1–2 wk
later, it is logical to look for effects of PTX on early events in the
immune response that coincide temporally with presence of PTX
in the system. Our previous work on the effect of PTX on Th1/Th2
phenotype commitment of effector T cells in animals immunized
for EAU induction indeed showed that both in mice and in rats,
PTX treatment strongly enhanced Th1 responses to the immunizing Ag in parallel to enhancing disease scores (21, 28). More recent data from other laboratories confirmed this and suggested that
activation of Ag-presenting cells and induction of IL-12 production are involved in this process (Ref. 29 and T. Forsthuber, unpublished observations). These results are in line with the early
observations of Lando et al. (27) describing effects on the Ag sensitization phase.
The second hypothesis, which is specifically examined in the
present study, was that if enhancement of vascular permeability is
indeed a major mechanism, the effect should be strongest if PTX
is administered at the time of effector cell migration. To our surprise, the data showed that PTX administered to animals coincident with effector cell trafficking and extravasation into the target
organ, aborts development of disease. The same is true if the eliciting Ag-specific T cells are pretreated with PTX before their infusion into animals. The phenomenon is dependent on the ADPribosyltransferase activity of PTX, indicating that Gi proteins are a
necessary target, and is accompanied by compromised responses to
chemokines of the affected lymphoid cells. Because migration, adhesion, and extravasation of leukocytes is highly dependent on
chemokine signals (24, 25, 30), the data are consistent with the
interpretation that prevention of EAU involves at least in part the
disruption by PTX of effector cell migration and infiltration into
the target organ, secondary to blockade of chemokine signaling
through Gi protein-coupled receptors. Our data also are consistent
with a recent study by Blankenhorn et al. (31) who demonstrated
(in mice concordant for MHC and for the locus controlling histamine sensitization) a single locus on chromosome 9 that controls
EAE severity and monocyte infiltration into the spinal cord. This
locus (eae9) includes a number of genes, among them the chemokine receptor CXCR5. The same group also presented evidence
that the locus eae7 on mouse chromosome 11 contains genes encoding the chemokines TCA3, monocyte chemoattractant protein
(MCP)-1, and MCP-5 (32).
The inhibitory effect of PTX administered to mice at different
times after transfer of uveitogenic T cells sheds new light on the
cellular events involved in EAU pathogenesis. Our previous study
(26) indicated that cellular infiltration into the eye during adoptively transferred EAU occurs in two discrete phases: 1) within
24 h of transfer, small numbers of activated uveitogenic T cells
enter the intact eye, apparently at random, and disappear by 72 h;
and 2) at 96 h, there begins a massive influx of host-derived bloodborne leukocytes (and additional Ag-specific T cells) that is
quickly followed by appearance of pathology. The result that PTX
injected either at 0 or 3 days inhibits disease indicates that EAU
can be prevented by interfering with either the first or the second
wave of cellular infiltration. The ability to prevent disease by inhibiting the second wave of infiltration alone supports the hypothesis that the Ag-specific T cells cannot by themselves cause pathology, and that tissue damage is dependent on the subsequently
recruited host effector cells. It is also in line with our previous
observation that recruited Ag-nonspecific T cells are required for
EAU pathogenesis, as athymic animals develop strongly attenuated disease after adoptive transfer of uveitogenic T cells (33). In
view of the critical importance of recruited host leukocytes for
EAU induction, it could be argued that treatment on day 0 prevented disease simply by inhibiting the secondary recruitment.
This possibility is argued against by experiments not shown here,
demonstrating that PTX prevented entry of fluorescently tagged
activated T cells into the retina at 24 h (S.B. Su et al., unpublished
results). Interestingly, although a single injection of PTX on day 0
(first wave of infiltration) was sufficient to abort disease, administration of PTX on day 3 (second wave of infiltration) appeared to
require a second dose several days later. We propose that the activated Ag-specific T cells must be allowed to enter the eye within
a very brief period of time after adoptive transfer or (in the absence
of continued stimulation) they will revert to a resting state in which
they are unable to induce disease (Ref. 34 and R. Caspi, unpublished results). Once the uveitogenic T cells have entered the eye
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FIGURE 7. Inhibition of infiltration into the eye of either the first wave
(Ag-specific T cells) or the second wave (recruited leukocytes) aborts development of EAU. B10.RIII mice were given 2 ⫻ 106 cells from a uveitogenic T cell line i.p. The mice received 0.5 ␮g of PTX i.v. at the indicated
times relative to adoptive transfer. EAU score was assessed 10 days later
by histopathology. The EAU scores of PTX-treated mice at day 0 or day
⫹3, ⫹7 (but not day ⫹3) vs nontreated group are significantly different
(p ⬍ 0.05).
INHIBITION OF EAU BY PTX
The Journal of Immunology
for inhibition of EAU, is long lived. It stands to reason that the
same may be true for other cellular effects of PTX, possibly including histamine sensitization of endothelial cells that affects cellular permeability. Thus, our data suggest that the effect of PTX on
autoimmune disease combines inhibitory as well as enhancing influences, with one or the other predominating at different stages.
The net observed outcome would thus represent an integration of
these opposing effects.
Acknowledgments
We are grateful to Drs. Joost Oppenheim and Ji Ming Wang, Frederick
Cancer Research Facility, National Cancer Institute, for their helpful critique of the manuscript.
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and initiated inflammatory changes, including chemokine production by ocular cells, a second dose of PTX is needed to prevent
disease development.
Our data do not address the question whether interference by
PTX with the first wave vs the second wave of cellular infiltration
into the eye involves the same mechanism(s). The chemokines
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inflamed tissue is dependent on chemokines to trigger adhesion of
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Our findings appear to be at variance with the well-documented
enhancing effects of PTX on autoimmunity. In EAE, PTX is
needed not only for disease induced by active immunization, but
also in many cases for disease induced by adoptive transfer of
primed T cells. How then, is EAU different in this aspect from
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INHIBITION OF EAU BY PTX