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Genetic Analysis of the Influence of Pertussis
Toxin on Experimental Allergic
Encephalomyelitis Susceptibility: An
Environmental Agent Can Override Genetic
Checkpoints
Elizabeth P. Blankenhorn, Russell J. Butterfield, Robert
Rigby, Laura Cort, Dana Giambrone, Paul McDermott, Kay
McEntee, Nancy Solowski, Nathan D. Meeker, James F.
Zachary, Rebecca W. Doerge and Cory Teuscher
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2000; 164:3420-3425; ;
doi: 10.4049/jimmunol.164.6.3420
http://www.jimmunol.org/content/164/6/3420
Genetic Analysis of the Influence of Pertussis Toxin on
Experimental Allergic Encephalomyelitis Susceptibility: An
Environmental Agent Can Override Genetic Checkpoints1
Elizabeth P. Blankenhorn,2* Russell J. Butterfield,† Robert Rigby,* Laura Cort,*
Dana Giambrone,* Paul McDermott,* Kay McEntee,* Nancy Solowski,* Nathan D. Meeker,†
James F. Zachary,† Rebecca W. Doerge,‡ and Cory Teuscher†
T
he use of adjuvants is a long-standing and often necessary
technique to induce autoimmunity in animals (1). Pertussis toxin (PTX)3 is an example of a potent ancillary adjuvant used to elicit several different autoimmune diseases, including experimental allergic encephalomyelitis (EAE), experimental
allergic orchitis, and experimental autoimmune uveitis (2– 4). Numerous theories have been proposed to explain the action of PTX
in the development of organ-specific autoimmune disease (5). The
prevailing hypothesis is that PTX “opens” blood-tissue barriers,
allowing enhanced infiltration of the T cells and macrophages that
are responsible for inflammation (5). This proposed role for PTX
is supported by EAE studies in which PTX injection leads to
breakdown of the blood-brain barrier (BBB) as revealed by the
influx of radioactive tracers into the brain parenchyma (6 – 8). In a
transgenic mouse model, pathogenic T cells accumulate in the
CNS only when PTX is administered (9). However, other wellknown activities of PTX, including its immunomodulatory, lymphocytosis-promoting, thymolytic, and lymphostatic effects, may
also contribute to disease pathogenesis (5, 10, 11).
*Department of Microbiology and Immunology, MCP Hahnemann University, Philadelphia, PA 19129; †Department of Microbiology and Immunology, School of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802; and
‡
Department of Statistics, Purdue University, West Lafayette, IN 47907
Received for publication October 28, 1999. Accepted for publication January 5, 2000.
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
This work was supported by National Institutes of Health Grants NS36526 (to C.T.
and E.P.B.), AI41747 (to C.T.), and NS25519 (to E.P.B.) and by National Multiple
Sclerosis Society Grants RG2659 and PP0625 (to C.T.).
2
Address correspondence and reprint requests to Dr. Elizabeth P. Blankenhorn, Department of Microbiology and Immunology, MCP Hahnemann University, 2900
Queen Lane, Philadelphia, PA 19129. E-mail address: [email protected]
3
Abbreviations used in this paper: PTX, pertussis toxin; EAE, experimental allergic
encephalomyelitis; BBB, blood-brain barrier; SC, spinal cord; SCH, SC homogenate;
QTL, quantitative trait locus; LRT, likelihood ratio test.
Copyright © 2000 by The American Association of Immunologists
As an initial step in understanding the genetic control of PTX
effects in regulating EAE susceptibility, we mapped the locus
(Bphs) controlling PTX-induced hypersensitivity to histamine
(12). This phenotype has been reported to be associated with susceptibility to EAE (13) and to experimental allergic orchitis (14).
Bphs is on chromosome 6 (12) and has been physically located
within a yeast artificial chromosome contig encoding Eno2,
Tnfrsf7 (Cd27), Ltbr, Tnfrsf1a (Tnfr1), and Vwf (15). However,
Bphs does not determine EAE susceptibility in all PTX-dependent
strains of mice, and some strains of mice differ in EAE susceptibility but do not differ in histamine sensitivity or Bphs alleles.
Therefore, additional loci controlling PTX effects must exist. The
purpose of the present study was to examine the genetic control of
PTX effects in EAE by using strains of mice that are concordant
for both Bphs and H2. Examination of EAE in (B10.S/DvTe ⫻
SJL/J) ⫻ B10.S/DvTe backcross (BC1) mice conditioned with
PTX allows the determination of whether PTX elicits more frequent and/or more severe clinical signs of EAE and the identification of additional loci mediating its effects. We report here that
the use of PTX significantly increases the proportion of backcross
mice with clinical signs of EAE. One major locus on chromosome
9 controlling PTX effects in EAE susceptibility was found, in contrast to the multiple eae-m loci that are involved in EAE in the
absence of PTX. Our results support the hypothesis that PTX is
capable of circumventing or overriding many of the genetically
determined checkpoints associated with EAE in this strain
combination.
Materials and Methods
Animals and immunizations
(B10.S/DvTe ⫻ SJL/J) ⫻ B10.S/DvTe backcross mice (BC1) were generated continuously over 12 mo from the same pool of B10.S/DvTe and
(B10.S/DvTe ⫻ SJL/J) F1 hybrid breeding stock. Inoculation of groups of
mice, ranging in age from 6 to 12 wk, was staggered over the same period.
Mice were injected s.c. at two sites on the posterior flank (0.15 ml/injection
site) with 1.0 mg of SJL/J spinal cord homogenate (SCH), in 0.15 ml PBS,
emulsified with an equal volume of CFA (16). A booster injection of SJL/J
0022-1767/00/$02.00
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Pertussis toxin (PTX) is a potent ancillary adjuvant used to elicit several different autoimmune diseases, including experimental
allergic encephalomyelitis (EAE). To delineate the genetics of PTX effect in EAE, we mapped EAE-modifying (eae-m) loci in
cohorts of backcross mice immunized with and without PTX. In this study, we analyzed the genetic basis of EAE susceptibility and
severity and the intermediate phenotypes of mononuclear cell infiltration, suppuration, and demyelination. In animals immunized
with PTX, one major locus, eae9, controls disease susceptibility and severity. Eae9 also regulates the extent of mononuclear cell
infiltration of the spinal cord in male mice. Without PTX, five eae-m loci were noted, including three new loci in intervals on
chromosomes 8 (eae14), 10 (eae17), and 18 (eae18). Taken together, these results suggest that eae9 controls the effects of PTX in
EAE susceptibility, and is capable of overriding the other genetic checkpoints in the pathogenesis of this disease. The Journal of
Immunology, 2000, 164: 3420 –3425.
The Journal of Immunology
3421
Table I. Incidence of clinical disease in PTX⫹ and PTX⫺ backcross mice
Group
Unaffected
Affected
% Affecteda
Day Onsetb
Average Peak Scorec
PTX⫹
Total
Male
Female
28
20
8
62
37
25
69
65
76
17.3 ⫾ 2.5
16.9 ⫾ 2.4
18.0 ⫾ 2.4
1.6 ⫾ 0.5
1.7 ⫾ 0.6
1.4 ⫾ 0.4
PTX⫺
Total
Male
Female
69
38
31
53
29
24
43
43
44
19.3 ⫾ 2.9
18.9 ⫾ 2.3
19.9 ⫾ 3.4
1.9 ⫾ 0.5
1.8 ⫾ 0.5
1.9 ⫾ 0.5
PTX⫹ vs PTX⫺: total, p ⬍ 0.0001; males, p ⫽ 0.016; females, p ⫽ 0.03.
PTX⫹ vs PTX⫺: total, p ⬍ 0.0001; males, p ⫽ 0.002; females, p ⫽ 0.03.
c
PTX⫹ vs PTX⫺: total, p ⫽ 0.002; males, p ⫽ 0.3; females, p ⬍ 0.0001.
a
b
Histological evaluation
Brain and SC samples were dissected from calvaria and vertebral columns,
respectively, and fixed by immersion in 10% phosphate-buffered Formalin
(pH 7.2) at 4°C. Following adequate fixation, brain and SC were trimmed
and representative transverse sections embedded in paraffin, sectioned at 5
␮m, and mounted on glass slides. Sections were stained with hematoxylin
and eosin for routine evaluation and luxol fast blue-periodic acid Schiff
reagent for demyelination. Representative areas of the brain and SC, selected for histological evaluation, were chosen based on previous studies
(18, 19). These included brain stem, cerebrum, and the cervical, thoracic,
and lumbar segments of the SC. Scoring was based on a semiquantitative
assessment for the following individual variables: 1) severity of the lesion
as represented by each component of the histological assessment; 2) extent
and degree of myelin loss and tissue injury (swollen axon sheaths, swollen
axons, and reactive gliosis); 3) number of neutrophils/eosinophils comprising the inflammatory exudate; and 4) number of lymphocytes/macrophages
comprising the inflammatory exudate. A score was assigned to the entire
brain and to the entire SC based on a subjective numerical scale ranging
from 0 to 5. A score of zero indicates no lesion, myelin loss, or inflammatory response was present in the tissue evaluated. The remaining scores
were as follows: 1, minimal; 2, mild; 3, moderate; 4, marked; and 5, severe
lesions, myelin loss, or inflammatory response present in the tissue.
Genetic analysis
Genomic DNA was isolated from liver tissue by standard techniques and
used as template for PCR amplification using radiolabeled primers as described previously (16). Primers for microsatellite markers were purchased
from Research Genetics (Huntsville, AL). Alleles at these markers were
resolved on denaturing polyacrylamide gels and read from autoradiographic films directly into a MapManager QT database (20). Linkage maps
were generated for the PTX⫹ and PTX⫺ groups using MapManager QT.
Linkage of marker loci to disease susceptibility was tested by ␹2 in 2 ⫻ 2
contingency tables. Quantitative trait loci (QTL) were identified by interval
mapping using model 3 of the Zmapqtl program in QTL Cartographer
version 1.13 (21). Traits used for interval mapping included highest score
seen during the course of EAE, severity index, monocyte/lymphocyte infiltration, suppuration, and demyelination of brain or SC. Critical values for
declaration of significant linkage were determined by the permutation
threshold theory (22). For this experiment, 1000 permutations of the data
were generated to provide a sampling distribution of the ␹2 or the likelihood ratio test (LRT) statistics under the null hypothesis of no linkage.
Each permutation was done by randomly shuffling and reassigning trait
values among the BC1 animals, while holding the genotypic information
fixed. Linkage analysis was then done for each permuted data set, and the
distribution of the maximum test statistic from each of the 1000 permutations was used to set experimentwise significance thresholds. Linkages
were reported if the test statistic for a particular marker locus was greater
than the experimentwise critical value calculated for ␣ ⫽ 0.10 (suggestive)
or ␣ ⫽ 0.05 (significant) for that trait. Significant linkages, or suggestive
linkages that replicate the findings of a previous study, were given eae
locus designations.
Interaction between marker loci
To test the hypothesis that significant QTL interact, we used a regression
model containing significant marker loci and two-locus interaction variables between significant marker loci. Regression models with dummy
coding for marker loci were analyzed in SAS using PROC REG (23).
Significance of the overall model was assessed using an F statistic, and
significance of interaction variables was assessed using a T statistic (24).
Results
Incidence of EAE in the two treatment groups
Signs of EAE were more frequent, and the latency to onset was
significantly shorter, in both males and females in the PTX⫹ group
(69% affected) compared with the PTX⫺ group (43% affected)
(Table I). Although the incidence of disease was higher in PTX⫹
mice, there was no difference in susceptibility between males and
females within either group. However, disease as measured by
average peak score (or by a severity index, data not shown) was
less severe in the PTX⫹ group, especially in females (Table I). A
substantial cohort of mice in the PTX⫺ group developed neuropathology in the absence of detectable clinical signs. We have
recently termed this type of disease benign EAE (25). Benign EAE
was prominent in PTX⫺ mice: 40% clinically unaffected PTX⫺
mice showed monocytes and lymphocytes in their SCs without any
outward signs of EAE, whereas only 16% of unaffected PTX⫹
mice had this phenotype. This was not influenced by sex.
Neuropathology in the two treatment groups
SC and brain samples were obtained from and scored for the occurrence and degree of mononuclear infiltration, suppuration, and
demyelination. The character and distribution of CNS histopathological lesions observed in this study were consistent with those
reported in previous studies (18, 19, 25). Inflammatory responses
in susceptible animals ranged from those with a predominantly
neutrophilic response admixed with a smaller monocytic/lymphocytic component to those with a predominantly monocytic/lymphocytic response. CNS tissue response ranged from mice with no
tissue injury to those with loss of myelin, reactive gliosis, swollen
axon sheaths, and swollen axons. In all mice with histopathology,
the inflammatory response had a perivascular distribution that was
predominantly observed in the meninges and in the white matter.
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SCH ⫹ CFA, prepared in the same manner as the primary inoculum, was
given on day 7. PTX-treated mice received 10 ␮g of crude PTX (17) i.p.
at the time of inoculation and 5 ␮g 24 – 48 h later. Ninety-two mice were
immunized using SCH, CFA, and PTX (PTX⫹) and 122 mice were immunized with SCH and CFA alone (PTX⫺). Mice were monitored daily for
symptoms and graded from 0 to 4 as follows: 0, no clinical expression of
disease; 1, floppy tail without hind limb weakness; 2, hind limb weakness
with or without flaccid tail; 3, hind leg paralysis and floppy tail; and 4, hind
leg paralysis accompanied by a floppy tail and urinary or fecal incontinence. Animals progressing to a score of 4 were euthanized. Animals were
observed for 30 or 60 days, at which time they were sacrificed and their
brains and spinal cord (SC) were retrieved for histological analysis. Severity of disease among affected animals was analyzed using a severity
index generated by averaging the clinical scores for each animal over the
number of days that it exhibited clinical symptoms. Liver tissue was collected for DNA isolation.
3422
PERTUSSIS TOXIN OVERRIDES GENETIC CHECKPOINTS IN MOUSE EAE
Table II. CNS histopathology in male and female PTX⫹ or PTX⫺ BC1 mice
PTX⫹
PTX⫺
Trait
Malea
Female
pb
Male
Female
SlC
Demyelination
Suppuration
Monocytic/lymphocytic infiltration
1.04
0.68
1.44
1.03
0.70
1.42
0.98
0.94
0.94
1.24
0.57
1.06
1.28
0.67
1.26
0.82
0.67
0.22
Brain
Demyelination
Suppuration
Monocytic/lymphocytic infiltration
0.15
0.47
1.16
0.09
0.33
1.51
0.34
0.33
0.03
0.41
0.49
0.91
0.83
0.84
1.52
0.0070
0.0100
0.0003
a
b
p
Mean value for the specified trait.
p value from Student’s t test comparing male and female mice.
Genetic control of EAE
Genome scanning was performed on both the PTX⫹ and PTX⫺
cohorts. We analyzed both the binary trait of EAE incidence (or
susceptibility) and the quantitative traits of severity and histopathology. For PTX⫹ mice, linkage to the binary trait of susceptibility was found in an interval that is marked by D9 Mit105 (␹2
values were 11.7 and 9.3, indicating probability of linkage at ␣ ⫽
0.05 and ␣ ⫽ 0.1, respectively). This effect came largely from
male mice (␹2 ⫽ 17.0). A QTL designated eae9 has previously
been identified in the same interval in an F2 intercross with the
same parental strains (16). In the PTX⫺ cohort, linkage to susceptibility was found to markers on chromosome 8, peaking at D8
Mit190 (␹2 ⫽ 18.6, ␣ ⫽ 0.1).
Loci that showed evidence of linkage to severity or histopathological signs of EAE are given in Table III. In the PTX⫹ cohort,
we again found linkage to a single major QTL (eae9) between D9
Mit22 and D9 Mit24. The SJL-derived allele at eae9 is associated
with increased severity. Stratification by sex revealed that one phenotype of eae9 is to increase monocyte/lymphocyte infiltration in
the SC in males. No linkage to any other locus, including previously identified eae-m loci, was seen in PTX⫹ mice. In contrast,
Table III. Quantitative trait loci linked to EAE susceptibility in PTX-treated vs untreated backcross mice
Treatment Group
PTX⫹
Total
Females
Males
PTX⫺
Total
Females
Males
LRT
Allelec
High score
Severity index
13.3a
15.1a
SJL
SJL
eae9
Mono/lymph SC
12.8b
SJL
eae14
eae14
eae14
eae7
eae7
eae14
eae14
eae14
eae17
eae17
eae11
eae18
eae18
High score
Severity index
Demyelination in SC
High score
Severity index
High score
Severity index
Demyelination in SC
High score
Severity index
Mono/lymph SC
Mono/lymph SC
Demyelination in SC
22.9a
25.9a
11.6b
12.8b
13.7a
19.8a
22.2a
13.3b
16.2a
15.8a
13.1a
17.8a
20.4a
B10.S
B10.S
B10.S
SJL
SJL
B10.S
B10.S
B10.S
SJL
SJL
SJL
SJL
SJL
Marker
cM
eae
D9Mit105
D9Mit105
None
D9Mit105
35
35
eae9
eae9
35
D8Mit190
D8Mit190
D8Mit190
D11Mit98
D11Mit98
D8Mit190
D8Mit190
D8Mit190
D10Mit31
D10Mit31
D16Mit12
D18Mit3
D18Mit3
21
21
21
58
58
21
21
21
36
36
27
54
54
Trait
a
LRT is significant for experimentwise threshold of ␣ ⬍ 0.05. Cutoffs for the ␣ ⫽ 0.05 level of significance range from LRT 12.9 to 15.6. The level
of significance indicated by an LRT ⬎15.6 was not established in this experiment.
b
LRT statistic given for each trait is significant for experimentwise threshold of ␣ ⫽ 0.1. Cutoffs for this level of significance range from LRT 11.5
to 12.5.
c
Indicates which parental allele of the EAE-modifying locus is associated with greater severity of clinical signs.
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In the SC, predilection for the nerve root entry zone was observed,
as reported previously (19).
When mice from the two treatment groups were examined for
the presence of histopathology in the CNS (brain or SC), the PTX⫹
group again had a higher incidence of affected mice than the PTX⫺
group for either CNS site (data not shown). Differences between
males and females were not observed for histopathological traits in
the brain or SC of PTX⫹ mice. In contrast, female mice in the
PTX⫺ group had significantly greater brain involvement than
males (Table II). Because animals were not all euthanized at the
same time points subsequent to the manifestation of their disease,
overall between-group comparisons could not be made for histological traits. However, we could analyze a subset of EAE-susceptible mice from each treatment group (51 PTX⫹ and 37 PTX⫺)
that were sacrificed within 1 wk of their last clinical sign, because
the lesions in these mice would be expected to be comparable.
Surprisingly, the extent of demyelination at either CNS site was
substantially less severe in the PTX⫹ than in the PTX⫺ subset (the
mean value for SC demyelination in these PTX⫹ mice was 1.39 ⫾
0.7 and for the PTX- mice was 2.32 ⫾ 0.63, p ⬍ 1 ⫻ 10⫺8; the
mean value for brain demyelination in PTX⫹ mice was 0.25 ⫾
0.44 and for the PTX⫺ subset was 0.89 ⫾ 0.88, p ⫽ 0.0002).
The Journal of Immunology
Multiple linear regression analysis
Multiple linear regression was used to test the hypothesis that significant QTL interact. Significant marker loci for disease severity
(D8 Mit190, D11 Mit98, and D10 Mit42), SC demyelination (D8
Mit190 and D18 Mit3), and SC monocytic/lymphocytic infiltration
(D8 Mit190, D16 Mit50, and D18 Mit3) were analyzed as independent variables in multiple linear regression analyses with the
appropriate phenotype as the dependent variable. To investigate
possible interactions between significant marker loci, two-locus
interaction terms were added to the regression models as independent variables. None of the interaction terms was significant ( p ⬎
0.05). Without interaction variables, statistical significance was
achieved for disease severity (F ⫽ 13.59, p ⬍ 0.0001), SC demyelination (F ⫽ 10.54, p ⬍ 0.0001), and SC monocytic/lymphocytic
infiltration (F ⫽ 6.31, p ⫽ 0.0005).
Discussion
In our study, EAE was more frequent in PTX-treated backcross
mice. However, disease symptoms were milder. These findings are
consistent with previous studies demonstrating that PTX can “convert” some EAE-resistant rodent strains to susceptible ones, indicating that autoimmune resistance is environmentally sensitive
(26). The ability to convert otherwise resistant mice implies that
some checkpoints in autoimmunity may be controlled by environmentally sensitive loci (27). For example, if a particular locus
maintains the integrity of the BBB, administration of PTX might
overcome that mechanism of EAE resistance. Thus, the activity of
any EAE resistance alleles at that locus could be bypassed by PTX
in a genetic cross where those alleles were segregating, and thus
linkage to that locus would be minimized. Characterization of the
loci controlling the activity of PTX in EAE will be helpful in
understanding the action of environmentally sensitive loci in susceptibility to autoimmune disease.
PTX has many effects on the immune system, aside from its
vasoactive amine-sensitizing activity (6, 7). Because our animals
were not segregating for the Bphs phenotype, which is believed to
be associated with BBB permeability changes, we expect that other
genetically regulated PTX-mediated phenotypes will be present in
our cross. The mechanism by which PTX affects EAE in this backcross could therefore be related to one of its other known activities
(5). For example, PTX causes a generalized lymphocytosis that is
accompanied by a depletion of lymphocytes in the spleen, lymph
node, and thymus due to the failure of cells to migrate back to the
peripheral lymphoid tissue (28, 29). PTX is also reported to be
preferentially mitogenic for T cells (30), and it has been reported
to enhance delayed-type hypersensitivity responses in an Ag-specific manner (31, 32). The enhanced delayed-type hypersensitivity
due to PTX correlates with elevated Ag-specific production of
IFN-␥ by these cells (33). Additionally, PTX can induce both Th1
and Th2 immune responses (34, 35) and can increase expression of
the costimulatory molecules B7-1 and B7-2 on macrophages and B
cells, and CD28 on T cells (35). In this regard, PTX is capable of
preventing the induction of peripheral T cell anergy to murine
encephalitogenic peptides, again in an Ag-specific manner (36);
central mechanisms of tolerance may also be affected by PTX (37).
Any of these or other known roles of PTX might be critical to the
development of clinical signs or neuropathology.
In the genetic analysis of the PTX⫹ group, only one locus (eae9)
shows evidence of linkage to disease susceptibility in comparison
to five loci in the PTX⫺ group. Our results therefore suggest that
PTX overcomes these critical genetic checkpoints in the etiology
of autoimmune disease. Eae9 was associated with the extent of
mononuclear infiltration in the SC and severity of EAE clinical
signs. This QTL is located in an interval encoding Blr1/CXCR5,
Il10ra ␣-chain, and Il18 (IFN-␥-inducing factor). Any of these
candidate genes could be responsible for the eae9-mediated phenotypes mentioned above. Given that PTX can induce increased
IFN-␥ production by T cells, it is interesting to consider the possibility that eae9 is Il18. Another possibility is that lymphocytosis
could be affected by the differential action of the chemokine receptor Blr1/CXCR5 alleles. This is particularly intriguing given
that the chemokines Scya1 (T cell activation 3), Scya2 (monocyte
chemoattractant protein (MCP)-1), and Scya12 (MCP-5) are candidates for eae7 on chromosome 11 (38), which was also identified
in the PTX⫺ backcross (Table III). In three separate crosses, therefore, we observed linkage to either chemokines or their receptors
(eae9 and eae7 in the BC PTX⫹ and PTX⫺, and eae7 in the F2 in
Ref. 38), and thus allelic differences in the chemokine-signaling
pathways may control susceptibility to EAE by regulating the migration of inflammatory cells and their access into the CNS.
The five QTL in the PTX⫺ cohort include a novel eae-m locus
we have named eae14. eae14 demonstrates significant linkage to
disease incidence and severity and to the extent of SC demyelination. This QTL maps to an interval containing several genes of
possible relevance to EAE, two of which are glutathione reductase
(Gr1) and caspase-3 (Casp3). Glutathione reductase (EC 1.6.4.2)
is an enzyme in the cytosol that maintains high levels of reduced
glutathione in the cell, which provides significant antioxidant activity. It is expressed in macrophages and in brain. A deficiency in
glutathione reductase in either of these tissues could render them
susceptible to the toxic effects of free oxygen radicals in the inflammatory site (39). C57BL strains have the GR1a allele and are
known to have lower enzyme activity, whereas SJL/J mice have
the Gr1b allele and higher enzymatic activity (40). This difference
in enzyme activity could explain why B/B homozygotes for eae14
develop more severe disease in the PTX⫺ group. The second candidate gene in this interval is caspase-3, a downstream effector
molecule of cellular apoptosis. The active form of caspase-3 is
highly expressed in the spleen and less so in the brain (41). It has
been hypothesized that apoptosis of T cells might play a role in the
resolution of EAE (42– 44).
The PTX⫺ cohort displayed sexual dimorphism. One linkage to
EAE severity was found on chromosome 10 only in PTX⫺ females. This interval contains several interesting candidate genes,
including matrix metalloproteinase 11 (Mmp11 at 41 cM), tissue
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five linkages were seen in the PTX⫺ cohort, including two QTL
previously shown to play a role in this strain combination (16, 25).
The most significant QTL in the PTX⫺ cohort is on chromosome
8 between D8 Mit3 and D8 Mit31. Susceptibility, severity, and SC
demyelination are associated with the B10.S-derived allele at this
locus, which we have designated eae14. In addition to eae14, a
locus on chromosome 11 that colocalizes with eae7 (16) contributed to severity in this cohort. As in the F2 intercross between these
same strains, sex-specific linkages were seen in the PTX⫺ mice.
Females from the PTX⫺ group showed linkage to chromosome 10
for severity of clinical signs in an interval between D10 Mit126
and D10 Mit10. We have designated this locus as eae17. A QTL
controlling severity of mononuclear infiltrates in the SC was
mapped to a broad region of chromosome 16. Increased severity of
inflammation was associated with inheritance of the SJL allele at
this locus, which was previously designated as eae11 (25). In
PTX⫺ males, a strong QTL was mapped to chromosome 18. The
SJL-derived allele at this locus (eae18) resulted in greater infiltration and demyelination in the SC. No QTL for brain histopathology met experimentwise cutoffs for suggestive (␣ ⫽ 0.10) or significant (␣ ⫽ 0.05) linkage in either cohort.
3423
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PERTUSSIS TOXIN OVERRIDES GENETIC CHECKPOINTS IN MOUSE EAE
Acknowledgments
We thank Bryan Wardell, Jayce Sudweeks, and Julie Teuscher for their
assistance on this project.
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inhibitor of metalloproteinase 3 (Timp3 at 49 cM), insulin-like
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pathogenesis. The precise mechanism of action of this gene remains unknown, but presumably reflects an intermediate phenotype other than PTX-induced vascular permeability changes. The
molecular characterization of this QTL will potentially aid in the
understanding of environment-gene interactions in autoimmune
disease.
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