By
From the * Keratinocyte Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United
Kingdom; and the Department of Histopathology, St. Mary's Hospital Medical School, Imperial
College of Science, Technology and Medicine, London WC2A 3PX, United Kingdom
Systemic lupus erythematosus (SLE) is a potentially fatal non-organ-specific autoimmune disease that predominantly affects women. Features of the disease include inflammatory skin lesions and widespread organ damage caused by deposition of anti-dsDNA autoantibodies. The
mechanism and site of production of these autoantibodies is unknown, but there is evidence
that interferon (IFN) plays a key role. We have used the involucrin promoter to overexpress
IFN-
in the suprabasal layers of transgenic mouse epidermis. There was no evidence of organ-specific autoimmunity, but transgenic animals produced autoantibodies against dsDNA and histones. Autoantibody levels in female mice were significantly higher than in male transgenic
mice. Furthermore, there was IgG deposition in the glomeruli of all female mice and histological evidence of severe proliferative glomerulonephritis in a proportion of these animals. Our
findings are consistent with a central role for the skin immune system, acting under the influence of IFN-
, in the pathogenesis of SLE.
Systemic lupus erythematosus (SLE) is a relatively common non-organ-specific autoimmune disease, with a
prevalence comparable to that of multiple sclerosis (1). SLE
predominantly affects women, the female/male ratio being
~9:1 (2). In this condition, splenomegaly and inflammatory skin lesions of varying severity occur in association
with autoantibody production against a variety of nuclear
antigens and multiple organ damage (1). Renal involvement complicates 60-70% of cases and its severity largely
determines prognosis (3). The renal glomeruli are the prime
site of injury due to the presence of anti-dsDNA autoantibodies in the mesangium and capillary walls (4). Anti-dsDNA
antibodies intravenously administered to mice have been
shown to induce glomerulonephritis (GN)1, although the
reason for tissue injury remains controversial (1, 4).
The mechanism of antinuclear autoantibody production
is unknown, but there is evidence that IFN- The site of production of pathogenic antinuclear autoantibodies in SLE is obscure. However, the observation that
UV radiation, a factor known to exacerbate the disease, can
induce translocation of nuclear antigens to the keratinocyte
surface suggests that the skin immune system may be involved (10, 11). We have recently made transgenic mice in
which IFN- Preparation of Transgenic Mice.
This was carried out as previously described (12). In brief, a transgene with the cDNA for
murine IFN- IFN- Indirect Immunofluorescence on Normal Mouse Skin and Esophagus.
Serum samples from transgenic mice and negative control
littermates were screened for the presence of autoantibodies by indirect immunofluorescence on normal mouse tail skin or esophagus. Unfixed, 8-µm-thick frozen sections of normal mouse skin/
esophagus were allowed to air-dry at room temperature for 30 min before staining. Sections were blocked for 30 min in PBS
containing 1 mM CaCl2, 1 mM MgCl2 (PBSABC), and 10% FCS
(Imperial Laboratories, Andover, UK) followed by incubation
with mouse serum at a dilution of 1:10 in PBSABC for 45 min.
Sections were then incubated for 45 min with Texas red-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Labs, Inc.,
West Grove, PA) at a dilution of 1:100 in PBSABC.
Cell Culture.
Isolation of human keratinocytes from newborn
foreskin and cultivation on a feeder layer of mitomycin C-treated
3T3 cells have been previously described (13, 14). The culture medium consisted of one part Ham's F12 medium and three parts
DMEM, supplemented with 1.8 × 10 Immunofluorescence Staining of Cultured Keratinocytes.
Mouse and
human keratinocytes grown on coverslips were fixed and permeabilized for 10 min using a 50:50 methanol/acetone solution at
Western Blotting.
Confluent cultured mouse and human keratinocytes were lysed on ice in extraction buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 0.1% Triton X-100, 2 mM PMSF, and 0.01% leupeptin. The lysates were
centrifuged at 14,000 rpm for 5 min and the pellets were discarded. The supernatants were subjected to SDS-PAGE on 6 or
8% slab gels, followed by transblotting to nitrocellulose paper.
Transblotting was carried out in 48 mM Tris, 387 mM glycine, and 3.5 mM SDS containing 20% methanol for 2 h at 35 V followed by 2 h at 70 V. The blots were blocked overnight in 5%
skim milk in PBS containing 0.01% Tween (PBS/T), then incubated for 1 h with serum from transgenic or control mice diluted
at 1:200 in PBS/T containing 0.1% BSA (Sigma Chemical Co.,
Poole, Dorset, UK). Nitrocellulose strips were then incubated for
1 h with horseradish peroxidase (HRP)-conjugated rabbit anti-
mouse Ig (DAKO, High Wycombe, UK) diluted 1:5,000 in PBS/T
containing 0.1% BSA. Between antibody incubations, strips were
washed three times with PBS/T. Additional strips were incubated
with rabbit antidesmoglein antibody 145 (raised against the repeat
region of desmoglein-1 and recognizing all three desmogleins, provided by Dr. Anthony Magee) followed by HRP-conjugated
donkey anti-rabbit Ig (Amersham International, Little Chalfont,
UK). All incubation and washing steps were performed at room
temperature. The peroxidase activity was visualized by enhanced
chemiluminescence (ECL; Amersham International).
Assay for Extractable Nuclear Antigens (ENA).
Serum samples
were screened for the presence of antibodies against extractable
nuclear antigens (Sm, U1RNP, SS-A [Ro], SS-B [La], Jo1, and
Scl-70) using a commercially available ENA screening counter
current immunoelectrophoresis (CIE) kit (The Binding Site, Ltd.,
Birmingham, UK). Transgenic serum found to be positive on this
screen was further characterized using an ENA typing CIE kit
specific for the same antigens (The Binding Site, Ltd.). For ENA
screening, 120 µl of ENA extract (buffered sheep spleen extract preserved in 100 mM PMSF and 10 mM mercaptoethanol) and
20 µl of test serum were applied to the surface of an agarose gel
by means of an application mask. For ENA typing, 15-20 µl of
test serum and 55-100 µl individual ENAs were impaired. For
both procedures electrophoresis was carried out at 50 V for 75 min on a Beckman Paragon power pack. Gels were stained in
Acid blue 29 in 5% vol/vol acetic acid for 2 s and destained in 5%
vol/vol acetic acid for 5 min. Gels were dried completely by
placing in a 45°C incudryer for 15 min and were then examined
for a visible immunoprecipitate.
Crithidia luciliae Staining.
Serum samples from transgenic and
negative control littermates were screened for the presence of
anti-dsDNA antibodies using a commercially available C. luciliae
dsDNA kit (The Binding Site, Ltd.). In brief, slides coated with
C. luciliae were incubated for 30 min at room temperature with
serum samples diluted 1:10 in PBS. The slides were washed in
PBS and then incubated for 30 min with FITC-conjugated goat
anti-mouse IgG (Jackson ImmunoResearch Labs.). After further
washing, the slides were mounted in Gelvatol (Monsanto Chemical Co., St. Louis, MO) and viewed using a confocal microscope.
Antihistone and Anti-dsDNA ELISA.
The levels of antihistone and anti-dsDNA antibodies in serum were measured using a
modification of previously described methods (16). Calf thymus
histones (Sigma Chemical Co.) were diluted in PBS to a concentration of 2.5 µg/ml and 0.2 ml of this antigen solution was
added to each well of an Immulon II microtiter plate (Dynatech
Labs., Inc., Chantilly, VA). After overnight incubation at 4°C, wells
were coated with 0.4 ml gelatin (1 mg/ml in PBS) for at least 24 h
at 4°C. After washing, 0.2 ml of serum samples diluted 1:1,000-
1:4,000 in 0.1% Tween, 1 mg/ml gelatin, and 0.5% BSA in PBS
were added and incubated for 1.5 h at room temperature. After
washing, HRP-conjugated rabbit anti-mouse Ig (DAKO) diluted 1:4,000 in 0.1% Tween in PBS was added. After 1.5 h of incubation at room temperature, the wells were washed and substrate
solution was added. The OD was then read with an automated
spectrophotometer at 492 nm.
plays a role.
First, the emergence of SLE, with de novo anti-dsDNA
antibody production, has been described in patients receiving systemic treatment with IFN-
or -
(5, 6). Second,
manipulation of the circulating amount and function of
IFN-
can profoundly alter the course of lupus in mouse
models of the disease; systemic administration of IFN-
accelerates the rate of progression to GN in lupus prone (NZB × NZW)F1 mice (7) and administration of anti-
IFN-
or soluble IFN-
receptor to these animals can delay development of the disease (7, 8). Nevertheless, the
precise effects of IFN-
on murine lupus appear to be critically dependent on the dosage of the cytokine, the timing
of administration, and the genetic background of the animal. For example, systemic administration of IFN-
to lupus prone MRL/lpr-lpr mice has no effect on the course of
the disease (9).
is expressed in the suprabasal layers of the
epidermis under the control of the involucrin promoter (12).
This results in marked overexpression of IFN-
in the epidermis but no increase in the level of IFN-
in the blood. IFN-
transgenic mice develop a nonblistering inflammatory skin disease with dermal edema and have marked splenomegaly. In addition, serum from the transgenic mice
contains antibodies that produce a nuclear pattern of staining on sections of normal mouse epidermis (12). In view of
these findings, we investigated IFN-
transgenic mice for
the presence of murine lupus.
under the control of the involucrin promoter was
injected into fertilized oocytes from (CBA × C57/BL10)F1 mice.
Three independent founder lines were generated: line 1205D
contains 2 copies of the transgene, 1205C contains 6 copies, and
1212F contains 32 copies. The characteristics of the mice which
were previously reported (12) and are reported here were observed in all three founder lines.
ELISA.
Serum from 15 transgenic mice (8 males, 7 females) aged 4-13 mo was tested for the presence of IFN-
using a murine IFN-
Cytoscreen immunoassay kit (Biosource
Intl., Camarillo, CA; reference 12). Recombinant mouse IFN-
(Genzyme, Cambridge, MA) in the range of 10-500 pg/ml was
used to standardize for known amounts of protein. In addition,
extracts of renal tissue from two 7-mo-old transgenic female mice
and two age- and sex-matched negative littermate controls were
prepared by sonication on ice in 1 ml PBS containing 0.2 mM
PMSF and 1 µM pepstatin. IFN-
levels in both kidneys from each animal were measured using the IFN-
Cytoscreen immunoassay kit and expressed per microgram of total protein present.
Experiments were performed in triplicate and chromogenic results were read on a spectrophotometer at OD450.
4 M adenine, 10% FCS,
0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 10
10 M cholera
toxin, and 10 ng/ml epidermal growth factor. An established mouse
keratinocyte line, provided by Dr. Rosario Romero (Imperial Cancer Research Fund) was grown under the same conditions as for human keratinocytes, except that the incubation temperature was 32 rather than 37°C.
20°C or in 3.7% formaldehyde for 10 min at room temperature, followed by 0.1% Triton X-100 in PBS for 5 min at room
temperature. The same staining patterns were observed with each
fixation technique. The coverslips were incubated with transgenic
mouse serum diluted 1:10 in PBSABC for 45 min. After washing
in PBS, coverslips were incubated with Texas red-conjugated
goat anti-mouse IgG for 45 min. In some experiments, coverslips
were incubated with rabbit antidesmoglein antibody 919 (recognizing the cytoplasmic repeat region of desmoglein-1 and -2, provided by Dr. Anthony Magee, National Institute for Medical
Research, London, UK; reference 15), followed by Texas red-
conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch
Labs.). All incubations and PBS washes were carried out at room
temperature. Stained cells were mounted in Gelvatol and examined using an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY).
Immunofluorescence Detection of Kidney Deposits. Kidney tissue from transgenic mice and negative control littermates was snap-frozen in an isopentane bath cooled in liquid nitrogen. Frozen sections embedded in OCT (Tissue TekTM; Miles Inc., Elkhart, IN) were cut at 5-8 µm thickness. Sections were air dried and blocked for 30 min with goat serum. Sections were then incubated for 45 min with Texas red-conjugated goat anti-mouse IgG diluted at 1:100. Each antibody incubation was carried out at room temperature and was followed by thorough washing in PBS. Stained sections were mounted in Gelvatol and examined using a Zeiss Axiophot microscope.
Histology and Electron Microscopy. For light microscopy, kidney tissue was fixed in formalin, paraffin-embedded, and sections were stained with hematoxylin and eosin, or periodic acid Schiff. For electron microscopy, kidney tissue was fixed in 4% buffered glutaraldehyde at 4°C, post-fixed in osmium tetroxide, and ultra thin sections were stained with uranyl acetate and Reynold's lead citrate.
34 transgenic animals and 12 littermate controls were examined. Tables 1 and 2 list the age
and sex of each animal and summarize several of the parameters measured. The majority of animals previously analyzed were under 5 mo of age (12). However, the mean
age of the transgenic mice and negative control littermates
in this study was greater; 8.1 and 9.6 mo, respectively. None
of the transgenic mice in the early study had detectable levels of circulating IFN- (12). We used the same ELISA
method to test the serum of 15 of the present group of
transgenic mice and 5 negative littermate controls (see Tables 1 and 2). All 5 controls and 11 of the transgenic animals had no detectable serum IFN-
, but 4 male transgenic mice had small amounts (15-40 pg/ml; limit of detection
in the assay is 5 pg/ml). We had previously reported that
IFN-
was readily detectable in skin of transgenic but not
control mice, the 1212F line having the highest concentration (33 pg/cm2; reference 12). We measured IFN-
levels
in kidney tissue extracts of two transgenic females from this
founder line (Nos. 33 and 34, Table 1) and two littermate
controls (C11 and C12, Table 2). The level of IFN-
detected was 4 pg/µg total protein in the transgenic mice and
6 pg/µg in the controls.
|
|
Previous studies in transgenic models
have shown that local overproduction of IFN- can result
in tissue-specific autoimmunity (17, 18). To test whether the
autoantibodies in our transgenic mice were keratinocyte-specific we stained sections of mouse esophagus and skin in
which keratinocytes, stromal fibroblasts, muscle cells, and
endothelial cells could all be identified. Positive staining of
all cell types was observed (Table 1 and data not shown). Serum from 14 out of 17 transgenic mice was positive. Serum from 6 out of 7 littermate controls was negative.
Autoantibodies directed against membrane antigens are a
feature of several autoimmune skin diseases, the antigens
frequently being proteins involved in cell-cell or cell-extracellular matrix adhesion (19, 20). The cellular distribution of
the antigens recognized by antibodies in the serum of the
IFN- transgenic mice was examined by staining cultured
mouse and human keratinocytes (Fig. 1, A and C). In the
10 serum samples examined (Table 1) there was intense
staining of the nucleus, with no evidence of membrane staining. Autoantibodies stained nuclei of both mouse and human cells. For comparison, keratinocytes were stained with an antibody to desmogleins, the autoantigens of pemphigus
vulgaris and pemphigus foliaceus; as illustrated in Fig. 1 B the
staining pattern was quite distinct from that observed with
autoantibodies from the IFN-
transgenic mice.
Antinuclear Antibodies in IFN-
To identify proteins recognized
by the autoantibodies from IFN- transgenic mice, extracts
of cultured mouse and human keratinocytes were resolved on
SDS-PAGE and subjected to immunoblotting (Table 1 and
data not shown). 10 of the 12 serum samples recognized a
single band with an apparent molecular mass of ~100 kD.
However, serum from two nontransgenic BALB/c control
mice also recognized the band, indicating that it was not
specific to the transgenic mice. As predicted from the immunofluorescence staining shown in Fig. 1, the 100-kD
band was not recognized by a pandesmoglein antibody (data
not shown). Furthermore, the mobility is distinct from that
of the bullous pemphigoid antigens (21).
Next, we tested the serum samples for antibodies to ENA using counter-current immunoelectrophoresis. Increased levels of autoantibodies against the ENA screened (Sm, U1RNP, SS-A [Ro], SS-B [La], Jo1, and Scl-70) were detected in one animal only (no. 16 in Table 1). On further characterization this mouse was found to be positive for anti-Sm autoantibodies.
To examine whether antibodies to dsDNA were present, we screened serum for reactivity to the kineoplast of the flagellate organism C. luciliae. Indirect immunofluorescence testing of serum on C. luciliae has been used as a specific test for the presence of anti-dsDNA autoantibodies (22). Serum samples from 21 transgenic mice and 12 negative littermate controls were tested. 18 samples from transgenic animals produced definite staining of the kinetoplast (see Fig. 2 A and Table 1). All littermate controls were negative on this test (Table 2).
We used ELISA assays to quantitate the levels of anti-dsDNA autoantibodies and to determine whether antihistone antibodies were also present. Sera from male (mice Nos. 1-9 and 19; mean age, 7.4 mo; Table 1) and female transgenic mice (mice Nos. 10-16; mean age, 8.6 mo; Table 1) and negative control littermates (mice C1-C6 for anti-dsDNA and mice C1-C4, C9, and C10 for antihistone, Table 2), were measured and are shown in Fig. 2, B and C and Table 3. Compared to littermate controls (mean OD, 0.038) both male (mean OD, 0.081; P <0.01) and female (mean OD, 0.232; P <0.003) transgenic mice showed evidence of anti-dsDNA antibody production. Levels were significantly higher in females than males (P <0.007). All four MRL/lpr mice, included as positive controls, produced higher levels of anti-dsDNA antibody than the transgenic females (mean OD, 0.848 vs. 0.232, respectively, Fig. 2 B). Female transgenic mice tested produced antihistone antibodies at levels comparable to MRL/lpr controls (mean OD, 0.313 and 0.478, respectively, Fig. 2 C). Antihistone antibody levels in serum from male transgenic mice did not differ significantly from negative littermate controls (mean OD, 0.011 and 0.013, respectively, Fig. 2 C). Antinuclear autoantibodies were detected in female animals from all three transgenic founder lines. Interestingly, mice that produced the highest levels of anti-dsDNA did not necessarily produce high levels of antihistone antibodies and vise versa (Table 3).
IFN-Anti-dsDNA antibodies are known to deposit in the kidneys of 60-70% of SLE patients and to cause
GN (3, 4). We therefore examined the kidneys of the transgenic mice for evidence of autoantibody deposition and
organ damage (Fig. 3). Immunohistochemistry of kidneys
showed dense deposits of IgG within the glomeruli in all female IFN- transgenic mice examined (n = 5, Table 1).
As in human lupus-nephritis, both mesangial (Fig. 3 A) and
capillary (Fig. 3 B) patterns of Ig deposition were found.
Five out of eight male mice tested had evidence of Ig deposits in the glomeruli (Table 1).
Histological examination of kidney tissue (16 males and 11 females) demonstrated clear evidence of GN in female mice only. The severity of the lesion varied from mild mesangial nephritis (results not shown) to severe diffuse proliferative GN (Fig. 3 C). The former was observed in mice from two founder lines, 1205C and 1212F, and overall occurred in ~25% of female animals examined (Nos. 12, 14, and 16, Tables 1 and 3). Subendothelial-mesangial deposits were confirmed by electron microscopy (Fig. 3 E) and the immunopathology closely corresponded with the findings in spontaneous murine lupus-like syndromes (23). Interestingly, severe proliferative GN occurred in female transgenic mice with high levels of anti-dsDNA (Table 3). Control littermates had normal kidneys (n = 10; Fig. 3 D).
We have shown that targeting an IFN- transgene to the
suprabasal epidermal layers via the involucrin promoter results in production of antihistone and anti-dsDNA autoantibodies and in immune complex deposition in the kidneys.
Autoantibody levels were higher in female mice than males
and kidney damage was only found in females. These observations, together with our earlier findings that the mice
had inflammatory skin lesions, dermal edema, and splenomegaly (12) suggest that our transgenic mice are a useful
model for SLE. Reevaluating the skin phenotype of the transgenic mice in the light of this interpretation, the occasional
separation of epidermis from dermis with infiltration of
hemopoietic cells is probably the hydropic degeneration of
basal cells that is characteristic of SLE (24).
The reason for the higher rate of SLE in females remains
unknown. However, the defects in experimental tolerance
which have been described in spontaneous murine lupus
are critically dependent on sex hormones, with androgens
exerting a protective effect (for review see reference 25). In
IFN- transgenic mice, the highest levels of both antihistone and anti-dsDNA autoantibodies were detected in female animals (Fig. 2, B and C). In addition, histological evidence of kidney pathology was only observed in female
transgenic mice. There was no correlation between antihistone levels and GN severity. Furthermore, while the two
female mice with highest levels of anti-dsDNA antibodies
had marked immune complex deposition in the glomeruli
(Table 1) and severe GN (Table 3), the correlation between anti-dsDNA levels and kidney damage was not absolute (compare mice 11 and 12, Table 3). This phenomenon is well described in patients with SLE. The extent of
tissue immune complex deposition, and the degree of organ damage, do not depend solely on the serum titer of anti-dsDNA antibodies but also on several qualitative properties
of the antibodies (26) and possibly on the host's ability to
process immune complexes (27).
Previous observations in transgenic mice in which IFN-
is overexpressed in specific tissues have suggested that local
overproduction of IFN-
is involved in the pathogenesis of
organ specific autoimmunity (17, 18). In contrast, our transgenic mice did not produce autoantibodies characteristic of
cutaneous autoimmune disease. Autoantibodies in our mice
reacted with multiple cell types on tissue sections and did
not recognize membrane proteins, including the autoantigens of pemphigus vulgaris or bullous pemphigoid, the two
major autoimmune skin disorders; instead, they recognized nuclear antigens. In the light of the earlier transgenic studies, the lack of organ-specific autoimmunity in our transgenic system may appear surprising. However, previous studies have strongly implied that the consequences of IFN-
overexpression may vary with tissue type. Transgenic expression of IFN-
in beta-cells results in a cell-mediated immune
destruction of pancreatic islets (17), whereas overexpression
in the neuromuscular junction elicits a humoral response
with no evidence of cell-mediated damage (18). It has been
argued that the response of APCs to cytokines may differ in
different tissue types, leading to different patterns of T cell
activation (28). Certainly, there is evidence that the skin
immune system has distinctive properties, both in terms of
its APC and keratinocyte functions (29) and these may explain the difference in response to IFN-
overexpression in
the skin compared to other transgenic model systems.
With the exception of one animal, increased levels of anti-dsDNA antibodies were not accompanied by production of autoantibodies against the ENA tested. Interestingly, the single mouse with anti-ENA antibodies was found to be positive for anti-Sm autoantibodies, a serological finding considered highly specific for SLE (30). However, the consistent generation of high levels of antihistone and anti-dsDNA antibodies in our transgenic mice in the absence of significant autoreactivity to ENA supports the concept that different pathogenic mechanisms underlie the generation of the two types of autoantibody (31).
There is strong evidence that presentation of nuclear antigens to CD4-positive T cells is involved in the production of pathogenic anti-dsDNA autoantibodies (1) and Desai-Mehta et al. have isolated and characterized a subset of
autoimmune T helper cells from patients with SLE (32) which
strongly induce anti-dsDNA antibody production. Immunological abnormalities in the skin of IFN- transgenic mice suggest two possible mechanisms of autoantigen presentation
to T cells. IFN-
transgenic mice demonstrate a marked alteration in Langerhans cell distribution, consistent with migration from the epidermis to the dermis and the draining
lymph nodes (12). These "professional" APCs could interact with autoreactive T cells in either site. However, transgenic mice also markedly upregulate keratinocyte MHC
class II and intracellular adhesion molecule-1 expression
and there is evidence of low level CD4-positive lymphocyte migration into the epidermis in some animals (12).
Therefore, it is possible that keratinocytes play the key role
in autoantigen presentation. It is known that IFN-
can induce translocation of nuclear antigens from the nucleus to
the cytoplasm in epithelial cells and that keratinocytes may
act as APCs under certain conditions (33, 34). Keratinocytes in IFN-
transgenic mice do not express the costimulatory molecule B7 (12). However, antigen presentation to T cells in the absence of costimulatory molecules
could trigger an aberrant autoreactive immune response
and, indeed, defective antigen presentation is a feature of
SLE (35).
Skin lesions are one of the classical clinical manifestations
of SLE. Our findings are consistent with a central role for IFN- and the skin immune system in the pathogenesis of
the systemic complications of the disease. Elucidation of the
precise mechanisms involved in the generation of antinuclear
autoantibodies by the skin immune system in these animals
may give valuable insights into the pathogenesis of SLE.
Address correspondence to Dr. Fiona M. Watt, Keratinocyte Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. Phone: 44-171-269-3528; FAX: 44-171-269-3078. Joseph M. Carroll's current address is Genetics Institute, Andover, MA 01810.
Received for publication 10 April 1997 and in revised form 14 July 1997.
J.P. Seery was supported by funds from Bristol-Myers Squibb and a European Union Biomed Network.We thank Anthony Magee and Nora Sarvetnick for reagents and Leonora Bishop and Simon Broad for helpful discussions and technical advice. We also thank Ronald Stokes and Karen Walker (Department of Immunology, Birmingham University, Birmingham, UK) for carrying out the ENA assay, and Jill Collar and the ICRF Electron Microscopy and Histopathology Units for technical assistance. We are grateful to Wendy Senior for typing the manuscript.
1. | Kotzin, B.L.. 1996. Systemic lupus erythematosus. Cell. 85: 303-306 [Medline]. |
2. | Whaley, K. 1992. Diseases of the immune system. In Muir's Textbook of Pathology. R.N.M. MacSween and K. Whaley, editors. Edward Arnold, London, UK. 204-252. |
3. | Appel, G.B., F.G. Silva, C.L. Pirani, and D. Estes. 1978. Renal involvement in systemic lupus erythematosus (SLE): a study of 56 patients emphasizing histological classification. Medicine (Baltimore). 57: 371-410 [Medline]. |
4. | Itoh, J., M. Nose, S. Takahashi, M. Ono, S. Terasaki, E. Kondoh, and M. Kyogoku. 1993. Induction of different types of glomerulonephritis by monoclonal antibodies derived from an MRL/lpr lupus mouse. Am. J. Pathol. 143: 1436-1443 [Abstract]. |
5. |
Ronnblom, L.E.,
G.V. Alm, and
K.E. Oberg.
1990.
Possible
induction of systemic lupus erythematosus by interferon-![]() |
6. |
Graninger, W.B.,
W. Hassfeld,
B.B. Pesau,
K.P. Machold,
C.C. Zielinski, and
J.S. Smolen.
1991.
Induction of systemic lupus erythematosus by interferon-![]() |
7. |
Jacob, C.O.,
P.H. van der Meide, and
H.O. McDevitt.
1987.
In vivo treatment of (NZB × NZW)F1 lupus-like nephritis
with monoclonal antibody to ![]() |
8. |
Ozmen, L.,
D. Roman,
M. Fountoulakis,
G. Schmid,
B. Ryffel, and
G. Garrotta.
1995.
Experimental therapy of systemic
lupus erythematosus: the treatment of NZB/W mice with
mouse soluble interferon-![]() |
9. |
Nicolleti, F.,
P. Meroni,
R. Di Marco,
W. Barcellini,
M.O. Borghi,
M. Gariglio,
A. Mattina,
S. Grasso, and
S. Landolfo.
1992.
In vivo treatment with a monoclonal antibody to interferon-![]() |
10. | Kawashima, T., E.G. Zappi, T.S. Lieu, and R.D. Sontheimer. 1994. Impact of ultraviolet radiation on the cellular expression of Ro/SS-A-autoantigenic polypeptides. Dermatology. 189(Suppl. 1):6-10. |
11. | Norris, D.A.. 1993. Pathomechanisms of photosensitive lupus erythematosus. J. Invest. Dermatol. 100: 58S-68S [Abstract]. |
12. |
Carroll, J.M.,
T. Crompton,
J.P. Seery, and
F.M. Watt.
1997.
Transgenic mice expressing IFN-![]() |
13. | Rheinwald, J.G. 1989. Methods for clonal growth and serial cultivation of normal human epidermal keratinocytes and mesothelial cells. In Cell Growth and Division. A Practical Approach. R. Baserga, editor. IRL Press, Oxford, UK. 81-94. |
14. | Watt, F.M. 1994. Cultivation of human epidermal keratinocytes with a 3T3 feeder layer. In Cell Biology: A Laboratory Handbook. J.E. Cells, editor. Academic Press, Inc., Orlando, FL. 1:83-89. |
15. | Wheeler, G.N., A.E. Parker, C.L. Thomas, P. Ataliotis, D. Poynter, J. Arnemann, A.J. Rutman, S.C. Pidsley, F.M. Watt, D.A. Rees, et al . 1991. Desmosomal glycoprotein DGI, a component of intercellular desmosome junctions, is related to the cadherin family of cell adhesion molecules. Proc. Natl. Acad. Sci. USA. 88: 4796-4800 [Abstract]. |
16. | Kotzin, B.L., and E. Palmer. 1987. The contribution of NZW genes to lupus-like disease in (NZB × NZW)F1 mice. J. Exp. Med. 165: 1237-1251 [Abstract]. |
17. |
Sarvetnick, N.,
J. Shizuru,
D. Liggitt,
L. Martin,
B. McIntyre,
A. Gregory,
T. Parslow, and
T. Stewart.
1990.
Loss of
pancreatic islet tolerance induced by ![]() ![]() |
18. |
Gu, D.,
L. Wogensen,
N.A. Calcutt,
C. Xia,
S. Zhu,
J.P. Merlie,
H.S. Fox,
J. Lindstrom,
H.C. Powell, and
N. Sarvetnick.
1995.
Myasthenia gravis-like syndrome induced by expression of interferon ![]() |
19. | Iwatsuki, K., H. Harada, J.Z. Zhang, K. Maruyama, and F. Kaneko. 1994. Regulation of pemphigus and desmosomal antigen expression by keratinocyte differentiation. Dermatology. 189 (Suppl. 1):67-71. |
20. | Liu, Z., L.A. Diaz, J.L. Troy, A.F. Taylor, D.J. Emery, J.A. Fairley, and G.J. Giudice. 1993. A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP 180. J. Clin. Invest. 92: 2480-2488 [Medline]. |
21. |
Labib, R.S.,
G.J. Anhalt,
H.P. Patel,
D.F. Mutasim, and
L.A. Diaz.
1986.
Molecular heterogeneity of bullous pemphigoid
antigens as detected by immunoblotting.
J. Immunol.
136:
1231-1235
|
22. | Sontheimer, R.D., and J.D. Gilliam. 1978. An immunofluorescence assay for double stranded DNA antibodies using the Crithidia luciliae kinetoplast as a double stranded DNA substrate. J. Lab. Clin. Med. 91: 550-558 [Medline]. |
23. | Andrews, B.S., R.A. Eisenberg, A.N. Theofilopoulos, S. Izui, C.B. Wilson, P.J. McConahey, E.D. Murphy, J.B. Roths, and F.J. Dixon. 1978. Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains. J. Exp. Med. 148: 1198-1215 [Abstract]. |
24. | Lever, W.F., and G.S. Lever. 1983. Connective tissue diseases. In Histopathology of the Skin. W.F. Lever and G.S. Lever, editors. J.B. Lippincott, Philadelphia, PA. 445-471. |
25. | Steinberg, A.D.. 1992. Concepts of pathogenesis of systemic lupus erythematosus. Clin. Immunol. Immunopathol. 63: 19-22 [Medline]. |
26. | Foster, M.H., B. Cizman, and M.P. Madaio. 1993. Nephritogenic autoantibodies in systemic lupus erythematosus: immunochemical properties, mechanisms of immune deposition, and genetic origins. Lab. Invest. 69: 494-507 [Medline]. |
27. | Duits, A.J., H. Bootsma, R.H.W.M. Derksen, P.E. Spronk, L. Kater, C.G.M. Kallenberg, P.J.A. Capel, N.A.C. Westerdaal, G.T. Spierenburg, F.H.J. Gmelig-Meyling, and J.G.J. van de Winkel. 1995. Skewed distribution of IgG Fc receptor IIa (CD32) polymorphism is associated with renal disease in systemic lupus erythematosus patients. Arthritis Rheum. 39: 1832-1836 . |
28. | Sarvetnick, N.. 1996. Mechanisms of cytokine-mediated localized immunoprotection. J. Exp. Med. 184: 1597-1600 [Medline]. |
29. | Bos, J.D., and M.L. Kapsenberg. 1993. The skin immune system: progress in cutaneous biology. Immunol. Today. 14: 75-78 [Medline]. |
30. | Tan, E.M.. 1989. Antinuclear antibodies: diagnostic markers for autoimmune disease and probes for cell biology. Adv. Immunol. 44: 93-151 [Medline]. |
31. | Ma, J., J. Xu, M.P. Madaio, Q. Peng, J. Zhang, I.S. Grewal, R.A. Flavell, and J. Craft. 1996. Autoimmune lpr/lpr mice deficient in CD40 ligand. J. Immunol. 157: 417-426 [Abstract]. |
32. | Desai-Mehta, A., C. Mao, S. Rajagopalan, T. Robinson, and S.K. Datta. 1995. Structure and specificity of T cell receptors expressed by potentially pathogenic anti-DNA autoantibody-inducing T cells in human lupus. J. Clin. Invest. 95: 531-541 [Medline]. |
33. | Baboonian, M., P.J. Venables, J. Booth, D.G. Williams, L.M. Roffe, and R.N. Maini. 1989. Virus infection induces redistribution and membrane localization of the nuclear antigen La (SS-B): a possible mechanism for autoimmunity. Clin. Exp. Immunol. 78: 454-459 [Medline]. |
34. | Nickoloff, B.J., L.A. Turka, R.S. Mitra, and F.O. Nestle. 1995. Direct and indirect control of T-cell activation by keratinocytes. J. Invest. Dermatol. 105: 25S-29S [Abstract]. |
35. | Garcia-Cozar, F.J., I.J. Molina, M.J. Cuadrado, M. Marubayashi, J. Pena, and M. Santamaria. 1996. Defective B7 expression on antigen-presenting cells underlying T cell activation abnormalities in systemic lupus erythematosus (SLE) patients. Clin. Exp. Immunol. 104: 72-79 [Medline]. |