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Deficiency in β2-Microglobulin, But Not CD1,
Accelerates Spontaneous Lupus Skin Disease
While Inhibiting Nephritis in MRL-Faslpr
Mice: An Example of Disease Regulation at
the Organ Level
Owen T. M. Chan, Vipin Paliwal, Jennifer M. McNiff, Se-Ho
Park, Albert Bendelac and Mark J. Shlomchik
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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 © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 167:2985-2990; ;
doi: 10.4049/jimmunol.167.5.2985
http://www.jimmunol.org/content/167/5/2985
Deficiency in ␤2-Microglobulin, But Not CD1, Accelerates
Spontaneous Lupus Skin Disease While Inhibiting Nephritis in
MRL-Faslpr Mice: An Example of Disease Regulation at the
Organ Level1
Owen T. M. Chan,* Vipin Paliwal,† Jennifer M. McNiff,‡ Se-Ho Park,§ Albert Bendelac,§ and
Mark J. Shlomchik2*†
M
RL-Faslpr mice (MRL/lpr)3 mice develop a spontaneous, lupus-like syndrome that affects a number of target organs. These mice develop glomerulonephritis,
interstitial nephritis, and vasculitis (1, 2). Cellular infiltrates occur
in the salivary glands and joints (3– 6). MRL/lpr mice also develop
spontaneous cutaneous lesions, resembling human discoid lupus
erythematosus, whereas other lupus murine models rarely develop
skin disease (7). Clinically, MRL/lpr mice experience hair loss and
scab formation, typically on the dorsal neck region and the ears.
Light microscopy demonstrates hyperkeratosis, acanthosis, hypergranulosis, liquefaction, dermal vasodilation, and dermal T cell
infiltration (7). Ig deposition also occurs along the dermal-epidermal junction (8). Cellular infiltrates include CD4⫹ and CD8⫹ T
cells as well as macrophages (9). Cutaneous lesions usually occur
after the onset of renal disease (7, 10) (O. T. M. Chan, J. McNiff,
and M. Shlomchik, manuscript in preparation).
Various lupus-prone mouse strains bearing targeted mutations
of genes important to immune function have generally demon-
*Section of Immunobiology, Departments of †Laboratory Medicine and ‡Dermatology and Pathology, Yale University School of Medicine, New Haven, CT 06520; and
§
Department of Molecular Biology, Princeton University, Princeton, NJ 08544
Received for publication January 4, 2001. Accepted for publication June 26, 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
This work was supported by National Institutes of Health Grants AR44077 (to
M.J.S.) and AI38339 (to A.B.) and was supported in part by a Pilot/Feasibility Award
from the Yale Skin Diseases Research Core Center, P30AR41942. O.T.M.C. was
supported by National Institutes of Health Training Grant AI07019.
2
Address correspondence and reprint requests to Dr. Mark J. Shlomchik, Department of
Laboratory Medicine, Yale University School of Medicine, 333 Cedar Street, P.O. Box
208035, New Haven, CT 06520-8035. E-mail address: [email protected]
Abbreviations used in this paper: MRL/lpr, MRL-Faslpr; ␤2m, ␤2-microglobulin;
␤2m⫺/⫺/lpr, ␤2m-deficient MRL/lpr mice.
3
Copyright © 2001 by The American Association of Immunologists
strated a uniform amelioration or exacerbation of autoimmune disease among the target organs studied (11). An example of uniform
disease down-regulation is the ␣␤ T cell-deficient MRL/lpr strain,
which has milder renal disease, delayed skin lesions, reduced lymphoaccumulation, and reduced Ig production (12, 13). Similarly,
IFN-␥-knockout MRL/lpr mice have ameliorated renal and salivary gland disease in addition to decreased lymphoaccumulation
and autoantibody production (14). Examples of uniform disease
up-regulation include MRL/lpr mice deficient in perforin, which
have increased kidney, liver, and salivary gland infiltrates (15), and
TNF receptor type I-deficient C57BL/6-Faslpr mice, which develop increased cellular infiltration in the kidney, liver, lung, and
knee joints (16).
These previous studies suggest that end-organ disease is an inevitable consequence of initial autoreactive cell activation and loss
of tolerance in secondary lymphoid tissues. Under this hypothesis,
for example, B cell autoantibody secretion would lead to deposition and damage in multiple target organs (i.e., kidney and skin),
after which a program of end-organ disease ensues in proportion to
Ig deposition. Observations we report here regarding ␤2-microglobulin (␤2m)-deficient MRL/lpr mice (␤2m⫺/⫺/lpr) differ from
the findings in these other knockout strains and suggest a model in
which local conditions in target organs can control disease
manifestations.
Christianson et al. (17) reported that certain aspects of autoimmunity were ameliorated in MRL/lpr mice deficient in ␤2m. They
noted that ␤2m⫺/⫺/lpr mice have only mild glomerulonephritis
and reduced numbers of CD3⫹CD4⫺CD8⫺B220⫹ lymph node T
cells. ␤2m⫺/⫺/lpr mice also have reduced total IgG1, rheumatoid
factor, anti-dsDNA, and anti-Smith. However, total IgM, IgG2a,
and IgG3 levels remained comparable to those of age-matched
MRL/lpr mice. Although renal disease was assessed in these mice,
no spontaneous skin disease was described.
0022-1767/01/$02.00
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When mutations that inactivate molecules that function in the immune system have been crossed to murine lupus strains, the result
has generally been a uniform up-regulation or down-regulation of autoimmune disease in the end organs. In the current work we
report an interesting dissociation of target organ disease in ␤2-microglobulin (␤2m)-deficient MRL-Faslpr (MRL/lpr) mice: lupus
skin lesions are accelerated, whereas nephritis is ameliorated. ␤2m deficiency affects the expression of classical and nonclassical
MHC molecules and thus prevents the normal development of CD8- as well as CD1-dependent NK1ⴙ T cells. To further define
the mechanism by which ␤2m deficiency accelerates skin disease, we studied CD1-deficient MRL/lpr mice. These mice do not have
accelerated skin disease, excluding a CD1 or NK1ⴙ T cell-dependent mechanism of ␤2m deficiency. The data indicate that the
regulation of systemic disease is not solely governed by regulation of initial activation of autoreactive lymphocytes in secondary
lymphoid tissue, as this is equally relevant to renal and skin diseases. Rather, regulation of autoimmunity can also occur at the
target organ level, explaining the divergence of disease in skin and kidney in ␤2m-deficient mice. The Journal of Immunology,
2001, 167: 2985–2990.
2986
␤2m DEFICIENCY ACCELERATES LUPUS SKIN DISEASE AND INHIBITS NEPHRITIS
Here we analyze skin disease in ␤2m⫺/⫺/lpr mice and report an
interesting dissociation in end-organ disease. ␤2m deficiency reduces kidney disease; however, the deficiency accelerates, rather
than suppresses, the onset of skin lesions. Thus, ␤2m⫺/⫺/lpr mice,
in having divergent disease in the kidney and skin, differ from
other knockout lupus models. ␤2m noncovalently associates with
classical and nonclassical MHC class I proteins and is required for
optimal expression of the protein complex (18). ␤2m deficiency
can affect several molecules and cellular compartments, prominently including CD8 T cells and CD1-dependent NK1⫹ T cells.
Therefore, we further investigated how ␤2m deficiency leads to
accelerated skin disease by studying CD1⫺/⫺ mice, which we
crossed onto the MRL/lpr background. These mice do not have
accelerated skin disease, indicating that ␤2m deficiency is not
working via regulatory NK T cells that depend on CD1 or on CD1
expression itself as on Langerhans or B cells. We discuss the implications of these findings for the pathogenesis of disease in target
organs.
Mice
Mice used in the study were progeny of brother-sister matings of homozygous
␤2m-deficient MRL/lpr mice obtained from The Jackson Laboratory (http://
jaxmice.jax.org/jaxmice-cgi/jaxmicedb.cgi?objtype ⫽ framedetail&stock ⫽
002453). The strain was derived from N10 crossed to MRL/lpr and thus has
99.9% MRL background genes. ␤2m-intact (i.e., wild-type) MRL/lpr mice
were obtained from The Jackson Laboratory (Bar Harbor, ME) or were B
cell-sufficient progeny derived during backcrossing of the hemizygous JHD
allele to MRL/lpr. These latter mice were from the N7 to N13 generation
and thus also had 99% MRL background genes. These mice were bred in
our specific pathogen-free animal colony at Yale University School of
Medicine (New Haven, CT).
MRL/lpr.CD1⫺/⫺ mice were derived by crossing the CD1-targeted allele (19) onto the MRL/lpr background. Homozygosity for lpr was fixed at
the first BC generation. To obtain homozygotes, mice were intercrossed at
either N6 (98.5% MRL genes) or N10 (99.9% MRL genes) and were typed
by PCR. Data obtained both cohorts of mice were pooled, as there were no
differences between them. These mice were bred initially at Princeton University, but were observed for skin disease as adults at Yale University,
housed under the same conditions as other mice in this study. Thus, all
mice were aged and followed for disease in the same animal housing room
at Yale.
Photography
Clinical observations were recorded using an EOS Rebel G camera (Canon,
Tokyo, Japan) with a 90-mm F2.8 macro lens (Sigma, Tokyo, Japan). The
camera and lens were mounted on a copy stand (model CS-2; Testrite,
Newark, NJ), while the subject lay below on the base. Photographs were
taken with Kodak Gold ASA 100 film (Eastman Kodak, Rochester, NY).
Renal disease grading
H&E-stained, formalin-fixed sections were graded as we have previously
described (20).
Skin disease grading
Skin samples from the shaved dorsal neck region were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin.
The severity of skin disease was graded based on a semiquantitative
scale using the following parameters: acanthosis: 1) mildly, 2) markedly, or
3) very markedly thickened dermis; hyperkeratosis: 1) mildly or 2) markedly increased amount of keratin; interface (liquefaction): 1) focal or 2)
extensive damage to basal cell layer; inflammation: 1) sparse or 2) heavy
infiltrates of dermal cells resembling lymphocytes; mast cells: numbers of
dermal mast cells counted at ⫻20 (0.5 mm) in five fields and averaged for
number of mast cells per 0.5 mm, measurements made sequentially in areas
of greatest histologic change; fibrosis: increased dermal cellularity with 1)
slight or 2) markedly thickened dermis; vessels: presence of dilated vessels
with hemorrhage, 1) focal or 2) diffuse; and ulcer: epidermal erosion or
ulcer recorded when present (0 or 1). The averages plus 1 SD were generated. For comparative purposes, all tissue sections were scored by one
observer (J.M.M.), who was blinded to their origin. Scores for MRL/lpr
Skin disease incidence and statistical analysis
Cohorts of ␤2m-deficient or CD1-deficient MRL/lpr mice and wild-type
MRL/lpr mice were followed in time, and the onset of macroscopic skin
disease was recorded along with the ages of the animals. Mice were considered affected when an area ⱖ0.5 cm of hair loss, ulceration, and induration typical of MRL/lpr skin disease (our unpublished observations) was
noted. All data was recorded in Microsoft Excel 98 (Microsoft, Redmond,
WA) for the Macintosh.
Survival-type curves of the mouse strains were plotted using the KaplanMeier method and were examined for significance with the Mantel-Cox
log-rank test using StatView 4.5 (Abacus Software, Berkeley, CA) for the
Macintosh. Values of p ⬍ 0.05 were considered significant.
Results
␤2m deficiency in MRL/lpr mice suppresses spontaneous lupus
nephritis
Glomerulonephritis was previously shown to be reduced in ␤2m⫺/⫺/
lpr mice as expected (17). We confirmed that a similar phenomenon was occurring in our mice, as expected, by formal scoring of
a small cohort; ␤2m⫺/⫺/lpr mice had lower scores for glomerulonephritis ( p ⫽ 0.049; Fig. 1, A and B) (17). In addition, interstitial
nephritis and vasculitis (17), which had not been previously evaluated, were reduced (Fig. 1, C and D). Inflammatory cellular infiltrates were noticeably decreased compared with those in agematched, diseased MRL/lpr controls. These data are summarized
in Fig. 1E. Thus, both Ab-mediated and cell-mediated diseases
were ameliorated. However, it should be noted that ␤2m-deficiency did not completely abrogate disease, because mild glomerulonephritis and cellular infiltrates were still present.
␤2m deficiency in MRL/lpr mice accelerates spontaneous lupus
skin disease
In contrast to the reduced renal disease, the kinetics of skin disease
onset as well as the penetrance of skin disease were dramatically
accelerated by ␤2m deficiency ( p ⬍ 0.0001; Fig. 2). A significant
difference emerged at 12–14 wk, when none of the ␤2m-intact
mice had skin disease, but one-third of ␤2m⫺/⫺/lpr mice already
had clinical disease. There was very little mortality in either cohort
at this age (data not shown), thus ruling out selective survival as a
reason for differences in skin disease incidence. The 50% incidence of disease was 15 wk of age for ␤2m-deficient mice and 31
wk for ␤2m-intact mice. By 28 wk of age, all ␤2m⫺/⫺/lpr mice
were affected, whereas, one-fourth of the original ␤2m-intact cohort remained unaffected at 44 wk, an age when most mice in the
␤2m-intact cohort had already succumbed to disease in other organs (data not shown). There was no significant difference between
males and females in either the ␤2m-intact ( p ⫽ 0.55) or ␤2mdeficient ( p ⫽ 0.55) cohorts.
It is unlikely that a foreign pathogen was responsible for initiating the onset of accelerated skin disease in the ␤2m⫺/⫺/lpr mice.
Lesions were never observed in our other nonautoimmunity-prone
strains housed in the same room. Furthermore, all the mice were
raised under specific pathogen-free conditions. It was also unlikely
that disease was caused by fighting, as we had numerous examples
of affected mice that were singly housed, and disease was similar
in males and females, whereas females are rarely observed to fight.
Finally, neither T nor B cell-deficient MRL/lpr mice, which are
more immunocompromised than ␤2m⫺/⫺/lpr mice, develop skin
disease despite being housed in the same colony, again strongly
arguing against a role for pathogen-induced lesions.
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Materials and Methods
mice lacking B cells (no disease control) were 0 for all parameters except
mast cells (4/0.5 mm; our unpublished observations, and O. T. M. Chan,
J. M. McNiff, and M. J. Shlomchik, manuscript in preparation). Similarly,
clinically unaffected skin from several mice was observed to be essentially
normal (data not shown).
The Journal of Immunology
2987
incidence of skin disease in MRL/lpr mice acquired from The
Jackson Laboratory (32 wk) and bred in our facility (27 wk) were
similar, with overall curves being statistically indistinguishable
( p ⫽ 0.40; data not shown). If anything, disease may have occurred faster in our BC mice than in mice acquired from Jackson,
but certainly not the reverse. These incidence rates are somewhat
slower than those reported by Furukawa and colleagues, most
likely due to different husbandry conditions, a factor thought to
affect the incidence of autoimmunity in MRL/lpr mice, as suggested by Furukawa and colleagues (21).
Pathological characteristics of lupus skin disease of ␤2m⫺/⫺/lpr
mice are similar to those of MRL/lpr mice
CD1-deficient mice have neither accelerated skin disease nor
ameliorated kidney disease
The expression of both classical MHC class I and CD1 molecules
is absent or markedly reduced in the absence of ␤2m; ␤2m-deficient mice are also deficient in CD8 and NK1⫹ T cells that depend
on these class I molecules (23–27). To distinguish whether ␤2m
As the ␤2m-intact control group included mice derived during
backcross of a JHD allele (although with 99% MRL background
genes), we considered that the incidence of skin disease may have
been affected in some unexpected way and that this could have
affected our conclusions. However, in our colony, the times to 50%
FIGURE 2. ␤2m deficiency accelerates the appearance of skin lesions in
MRL/lpr mice. Survival-type curves were generated as described in Materials and Methods using the Kaplan-Meier method and were examined
for statistical significance using the Mantel-Cox log-rank test. Spontaneous
skin lesions on ␤2m-deficient MRL/lpr mice developed earlier than on
␤2m-intact MRL/lpr mice (p ⬍ 0.0001). The 50% incidences of disease
were 15 wk of age for the ␤2m-deficient mice and 31 wk for the ␤2m-intact
mice. The sample sizes were: ␤2m⫺/⫺/lpr, n ⫽ 86; and MRL/lpr, n ⫽ 110.
FIGURE 3. ␤2m⫺/⫺/lpr mice have comparable spontaneous cutaneous
disease as MRL/lpr mice. Clinically, skin lesions formed primarily at the
dorsal neck region of ␤2m⫺/⫺/lpr (A) and MRL/lpr (B) mice. The ears also
experienced disease at times. Neck skin with clinical lesions was biopsied
and fixed in 10% formalin. Sections were stained with hematoxylin and
eosin. Light microscopy demonstrated that both ␤2m⫺/⫺/lpr (C) and MRL/
lpr (D) mice developed comparable histopathology. The ␤2m⫺/⫺/lpr mice
were 17 wk of age (A and C), and the MRL/lpr mice were 22 wk (B and
D). C and D, ⫻200.
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FIGURE 1. ␤2m deficiency inhibits nephritis in MRL/lpr mice. Kidneys
from ␤2m-deficient and ␤2m-intact mice were fixed in 10% formalin, and
sections were stained with H&E. Light microscopy revealed an amelioration of glomerulonephritis, interstitial nephritis, and vasculitis in ␤2m⫺/⫺/
lpr mice (A and C) compared with MRL/lpr mice (B and D). Although
overall renal disease was decreased in ␤2m⫺/⫺/lpr mice, there remained
some areas of focal cellular infiltration (C). E, Summary of histologic grading (20). Shown are the mean and SD. There were 10 mice in each group.
o, ␤2m⫺/⫺/lpr; 䡺, control ␤2m-intact mice. p values are: glomeruli, 0.049;
vessels, 0.012; tubules, 0.047. The mice were 22 wk of age. Magnification:
A and B, ⫻500; C and D, ⫻200.
Clinically, the skin lesions in ␤2m-deficient and ␤2m-intact mice
were similar (Fig. 3). Lesions were localized to the dorsal neck
region and ears and typically were not observed elsewhere. However, disease was usually more aggressive in the ␤2m-deficient
mice, which had more total skin area affected than the MRL/lpr
controls (Fig. 3). Histologically, lesions in the two types of mice
were indistinguishable. This was evaluated formally by the reading
of coded slides (see Materials and Methods). The data are summarized in Table I. Overall, disease most resembled discoid lupus
erythematosus lesions (22), with prominent acanthosis, hyperkeratosis, and interface change. This is consistent with previous reports (7); our data add to those from previous studies of MRL/lpr
skin disease by using a more systematic classification according to
criteria used to evaluate human discoid lupus erythematosus lesions. In addition, Ig was deposited in the dermis and along the
dermal-epidermal junction of both strains, as assayed by immunofluorescence (data not shown).
␤2m DEFICIENCY ACCELERATES LUPUS SKIN DISEASE AND INHIBITS NEPHRITIS
2988
Table I. Skin disease scores for MRL/lpr and ␤2m⫺/⫺/lpr micea
Range
Acanthosis
(0-3)
Hyperkeratosis
(0-2)
Interface
(0-2)
Inflammation
(0-2)
Mast Cells
(no. per 0.5 mm)
Fibrosis
(0-2)
Vessels
(0-2)
Ulcer
(0-1)
MRL/lpr (n ⫽ 10)
␤2m⫺/⫺/lpr (n ⫽ 8)
2.0 ⫾ 0.7
2.1 ⫾ 0.6
1.9 ⫾ 0.3
1.9 ⫾ 0.4
1.7 ⫾ 0.5
1.9 ⫾ 0.4
1.7 ⫾ 0.5
2.0 ⫾ 0.5
21 ⫾ 16
14 ⫾ 8
1.8 ⫾ 0.4
1.8 ⫾ 0.5
1.6 ⫾ 0.5
1.9 ⫾ 0.4
0.4 ⫾ 0.5
0.6 ⫾ 0.7
a
Average scores and one standard deviation are listed for each strain. Score ranges are listed in parentheses under each disease category, except for mast cells. Comparisons
of each category were not statistically different. Ages for the mice were: MRL/lpr, 16 – 48 wk, ␤2m⫺/⫺/lpr, 17–24 wk.
Discussion
The current study reports an interesting discordance in spontaneous end-organ disease. Nephritis is inhibited in ␤2m⫺/⫺/lpr mice,
whereas cutaneous disease is accelerated, rather than suppressed.
The ␤2m⫺/⫺/lpr strain is the first reported lupus model with exacerbated skin disease coupled with diminished renal disease.
Nearly all published knockout autoimmunity-prone strains demonstrated either a uniform up-regulation or down-regulation of
end-organ pathology (11). CD40L⫺/⫺ MRL/lpr mice have reduced
kidney disease (nephritis, vasculitis, and renal Ig deposition), like
␤2m⫺/⫺/lpr mice (13). Cutaneous lesions in CD40 ligand-deficient
mice are not inhibited and are comparable to those in MRL/lpr
controls. However, as far as can be discerned, skin disease is not
accelerated as it is in the ␤2m⫺/⫺/lpr strain. The phenotypes of
both the ␤2m⫺/⫺/lpr and CD40 ligand⫺/⫺ MRL/lpr strains support
the hypothesis that regulation of autoimmune pathogenesis, presumably at the level of the effector cell, occurs in individual target
organs.
A number of mechanisms exist that could potentially explain the
up-regulation of skin disease in the ␤2m⫺/⫺/lpr strain. For example, the presence of ␤2m-dependent molecules on the target tissue
might have suppressive effects on pathogenic effector cells (28).
The absence of these ␤2m-dependent proteins in ␤2m⫺/⫺/lpr mice
would prevent any such inhibition. Indeed, target cell lysis by NK
cells is inhibited upon interaction with classical MHC class I molecules (29 –33). However, this mechanism does not readily explain
why skin vs kidney would be more susceptible to immune cell
attack.
The MHC class I-related receptor, FcRB, plays a role in the
regulation of serum Ig levels, possibly by preventing the catabolism of IgG (34). Indeed, serum IgG has a short half-life in ␤2mdeficient mice (35–37). ␤2m⫺/⫺/lpr mice have a decrease in total
IgG1 (17), which could explain the ameliorated renal disease (Fig.
1) (17). However, this observation would not account for their
exacerbated skin disease (Fig. 2).
Another possibility is that the absence of ␤2m prevents the maturation of suppressive, regulatory cells. One candidate is the
NK1⫹ T cell, the development of which is dependent on the expression of CD1 (23–27), a ␤2m-associated molecule. NK1⫹ T
cells are important for the early production of IL-4, a Th2 cytokine
capable of down-regulating Th1 cytokines (25, 38 – 40), and lupus
pathogenesis has been argued to be augmented by Th1 cytokines
(41– 45). We constructed CD1-deficient MRL/lpr mice to directly
test this idea. However, we found that CD1 deficiency does not
contribute to the ␤2m-deficient phenotype of accelerated skin disease, excluding a role for NK1⫹ T cells and CD1 expression on
APCs and target tissue.
␥␦⫹ T cells also have the potential to suppress or regulate lupus.
Peng et al. (46) demonstrated that ␥␦⫹ T cell-deficient MRL/lpr
mice developed exacerbated autoimmune renal disease, suggesting
that such T cells play a regulatory role. Evidence that a subset of
␥␦⫹ T cells may be positively selected in development by MHC
class I molecules comes from transgenic mice bearing TCR␥␦
transgenes specific for MHC class I (47, 48). However, it should be
noted that ␥␦⫹ T cells do not generally require positive selection
via MHC class I, as shown by the detection of ␥␦⫹ T cells in the
thymus, secondary lymphoid organs, and epithelia of ␤2m-deficient mice (49 –51). Thus, the potential role of ␥␦⫹ T cells in
modulating lupus skin disease requires further investigation.
FIGURE 4. CD1 deficiency does not affect the appearance of skin lesions in MRL/lpr mice. Survival-type curves were generated as described
in Fig. 2. The sample sizes were: CD1⫺/⫺/lpr, n ⫽ 13; and CD1-sufficient
MRL/lpr, n ⫽ 36. There was no difference between the groups (p ⫽ 0.246).
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
deficiency mediated its effects on skin disease through lack of CD1
and/or CD1-dependent NK1⫹ T cells, we studied cohorts of CD1deficient mice. CD1-deficient MRL/lpr mice did not demonstrate
accelerated skin disease (Fig. 4; p ⫽ 0.246), as disease incidence
was equivalent to that in CD1-sufficient mice derived as littermates
of heterozygote intercrosses. Incidence in both CD1-sufficient and
-deficient mice was somewhat more accelerated than that in the
MRL/lpr control cohorts used to compare with the ␤2m-deficient
mice (see above). This could be due to the less backcrossed nature
of the CD1 cohorts. As these data were acquired after the ␤2mintact data, we cannot formally rule out a change in the overall rate
within the colony, although there were no specific changes made in
husbandry conditions during this time. Regardless, the internal
comparison between CD1-deficient and -sufficient mice was conducted in concert and showed no difference. Most importantly,
disease occurred more slowly in the CD1 cohort than in the ␤2mdeficient mice ( p ⫽ 0.0001), thus ruling out that CD1 plays a
major role in the ␤2m-deficient phenotype.
These data exclude a role for NK1⫹ T cells and CD1 expression
in controlling skin disease. Similarly, grading of nephritis and vasculitis in a small subset of CD1-deficient and -sufficient mice revealed equivalent, severe disease (data not shown). Again, the
CD1-deficient mice contrast with the ␤2m-deficient mice, in which
nephritis and vasculitis were markedly reduced (17). Thus, CD1
and NK1⫹ T cells do not play a role in the amelioration of kidney
disease observed in ␤2m-deficient mice.
The Journal of Immunology
Gene-mapping studies (72–74) should consider this possibility, as
it may be that distinct loci control disease in particular target
organs.
Acknowledgments
We thank Drs. Joseph Craft, Ann Haberman, and Robert Tigelaar for critically reading this manuscript.
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Finally, CD8⫹ T cells, whose maturation is dependent on MHC
class I in the thymus (50, 51), may also negatively regulate immune responses, for example by acting on T cells (52, 53), dendritic cells (54), B cells (55), or CD11b⫹ monocytes/macrophages
(56). In addition, CD8⫹ T cells regulate T cell responses through
nonclassical MHC class I molecules, such as Qa-1, which depend
on ␤2m for proper expression. CD8⫹ T cells are induced by recognition of Qa-1-Ag complexes (57, 58) and delete Qa-1⫹, CD4⫹
T cells in an Ag-restricted manner after superantigen administration. Interestingly, stimulation of CD8⫹ T cells via Qa-1 on activated B cells induced the production of IFN-␥ (52), which could
suppress Th2 cells (59) and IgM and IgG1 Ab responses (60, 61).
Given these defined regulatory roles of CD8⫹ T cells along with
the clear dependence of CD8⫹ T cell development on ␤2m, CD8⫹
T cells are strong candidates for regulating skin disease in our
model.
Although we were able to use CD1 knockout mice to rule out a
role for NK1⫹ T cells and CD1, maturation of a subset of ␥␦⫹ T
cells and CD8⫹ T cells is also affected by ␤2m deficiency. To
investigate the regulatory role of each cell type will require analysis of disease in each of the respective knockout mice. CD8⫺/⫺
MRL/lpr mice were reported to develop skin vasculitis (62). Unfortunately, the study did not characterize the kinetics of the cutaneous disease; whether skin lesion onset was accelerated is unknown. We were unable to establish successful breeding of
CD8⫺/⫺ MRL/lpr mice. This strain may be extinct and will need
to be remade. Characterization of spontaneous skin disease has
not been conducted in ␥␦⫹ T cell-deficient MRL/lpr mice. Qa-1
knockout mice, which would also be of interest, do not yet exist as
far as we are aware.
A key question raised by our findings in the ␤2m⫺/⫺/lpr mice is
why there is differential immunoregulation in skin and kidneys.
The most obvious answer is that skin is a barrier organ, whereas
kidney is not. Barrier sites, such as skin, gut, and respiratory tract,
have specialized immune systems (63– 65). These systems are designed to respond efficiently to breach of the barrier, but are also
prone to inflammatory diseases, such as asthma, inflammatory
bowel diseases, lupus, and graft-vs-host disease. Perhaps to prevent overly exuberant responses, these local immune systems have
a variety of embedded, regulatory mechanisms as well. For example, immune responses in the lung may be preferentially deviated
toward Th2-type responses (66 – 68). In the skin and gut, ␥␦⫹ T
cells may play a regulatory role (69, 70). A similar role is possible
for other lymphocytes, as discussed above. The most likely interpretation of our data is that a ␤2m-dependent, regulatory cell operates in skin, but not in the kidney. Thus, in skin the natural
proinflammatory tendency, presumably not found in the nonbarrier
organs (i.e., kidney), may be unopposed in the absence of ␤2m.
If this concept is correct, it would fit with a modified view of
pathogenesis for systemic autoimmunity. In this view, regulation
of autoreactive cells can occur both at the stage of initial activation
that presumably occurs in secondary lymphoid tissues and in the
target tissues themselves, when autoreactive lymphocytes have become or are differentiating into effector cells. Our results showing
divergent effects on disease in kidney and skin are best accounted
for by this model. In nearly all other cases, mutations inactivating
molecules or cells in the immune system have had concordant
effects, either ameliorating or exacerbating disease in all target
organs (11). These studies had led to the concept that loss of tolerance resulted in an inevitable program of target organ pathology
(71). It is interesting to speculate that the spectrum of affected
organs, which differs among lupus patients, is in part due to genetically based variation in defects in organ-level immune regulation, such as manifested in the ␤2m-deficient model studied here.
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