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Departments of
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Immunology and
Rheumatology, Mayo Clinic and Medical School, Rochester, MN 55905
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several hypotheses have been proposed to explain the role of enterobacteria in HLA-B27-associated diseases including: 1) molecular mimicry of HLA-B27 with enterobacteria, 2) presentation of arthritogenic peptide from environmental Ag by HLA-B27 to CD8+ T cells, and 3) presentation of exogenous Ag by empty HLA-B27 (8, 9). More recently, a few investigators have proposed a role for B27-derived peptides in the disease. Sequence analysis of hypervariable regions of HLA-B27 with disease-implicated enterobacteria with B27-binding motif showed that a number of peptides fulfill this criteria and could be important in the initiation of autoimmune response (10). Binding of such peptides with HLA-B27 has been demonstrated in an in vitro assembly assay (11). On the other hand, HLA-B27-derived peptides may also be presented by MHC class II molecules (12, 13).
The role of CD8+ T cells vs CD4+ T cells in B27-linked diseases has been a controversial issue. Even though the HLA-B27 molecule as a class I Ag would be expected to present Ags to CD8+ T cells, most of the reports describe CD4+ T cells in the diseased joint or an MHC class II-restricted immune response against bacteria implicated in spondyloarthropathies (14, 15, 16). Only a few reports describe isolation of bacteria-specific B27-restricted CD8+ T cells from the involved joints of patients with B27-associated reactive arthritis and ankylosing spondylitis (17, 18). This further suggests the involvement of a CD4+ T cell recognizing a B27-derived peptide presented by class II molecules (13, 19).
We have reported an animal model of B27-associated arthropathy in which ß2m3-deficient HLA-B27 mice develop disease after they are transferred from a barrier facility to the conventional colony (4). These mice have low cell surface expression of B27 heavy chains (HC) and normal expression of endogenous class II molecules. In this mouse model of human disease, two possibilities may exist for the role of B27 in the disease process: 1) presentation of exogenous environmental peptide by ß2m-free HLA-B27 HC; 2) presentation of B27-derived peptides by endogenous MHC class II molecules. If the presentation of B27-derived self peptide by mouse H2-A molecule is related to disease development, MHC class II-deficient B27+ß2moAßo mice should not develop spontaneous disease. On the contrary, B27+ß2moAßo mice developed spontaneous disease similar to the previously described disease in B27+ß2mo mice, suggesting that class II molecules are not required for disease development. Furthermore, in vivo treatment with HC-specific HC10 Ab decreased the incidence of spontaneous disease in B27+ß2mo mice. Thus, blocking of B27 HC may prevent disease in experimental animals. Our data suggest a direct involvement of HLA-B27 HC as an Ag-presenting molecule, and not as an autoantigen in B27-associated disease in transgenic mice. Likewise, B27-linked human spondyloarthropathies may also be the result of direct function by B27 HC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of
B27+ß2mo mice has been
described before (4). ß2moAßo
mice and B27+Aßo mice were produced by mating
ß2mo mice and B27+ mice with
Aßo mice, respectively (20) (kindly provided by Drs. C.
Benoist and D. Mathis, INSERM, Strasbaurg, France). To generate MHC
class II-deficient B27+ß2mo mice
(B27+ß2moAßo),
ß2moAßo mice were mated with
B27+Aßo mice, and in the F2
population,
B27+ß2moAßo mice
were identified and intercrossed to generate the B27+ and
B27- mice in the context of ß2mo
and Aßo mutant genes. A flow chart describing the
generation of
B27+ß2moAßo mice is
shown in Figure 2
. All breedings were conducted in the barrier facility
of the Immunogenetics mouse colony of Mayo Clinic (Rochester,
MN).
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In the absence of ß2m, MHC class I molecules are rarely expressed on cell surface. Therefore, the presence of MHC class I transgenes in ß2mo mice was analyzed by PCR. DNA was extracted from the peripheral blood according to manufacturer’s instructions using the Isoquick nucleic acid extraction kit. Four microliters of DNA were added to 0.2 mM dNTPs, 1 µM each 3' and 5' primers in the PCR buffer in a total volume of 25 µl. A quantity amounting to 0.625 U of Taq polymerase was added to this mixture and amplified in 30 cycles under the following conditions: 3 min at 94°C (94°C for 1 min, annealing temperature 62°C for 1 min, and 72°C for 1 min), x 30, and 7 min at 72°C. PCR products were analyzed by electrophoresis, and their m.w. was compared with a standard m.w. marker.
Presence of HLA-B27 transgene was identified by PCR using the following oligonucleotides: 3'-(CTC TGC CTT GGC CTT GCA GA) and 5'-(CCA CTC CAT GAG GTA TTT CCA).
The presence of homozygous mutation in mouse ß2m was identified by PCR (5). We used three primers to identify the presence of wild-type and mutated mouse ß2m gene. Mß2m-1 and Mß2m-2 oligonucleotide sequences amplify wild-type ß2m. Neomycin and Mß2m-1 primer sequences amplify mutated ß2m. Presence of both of the PCR products indicates heterozygocity for the knockout gene. Mß2m-l: 3'-(GAA AAC CCC TCA AAT TCA AGT ATA CTC A). Mß2m-2: 5'-(GAC GGT CTT GGG CTC GGC CAT ACT). Neomycin: 5'-(TCG AAT TCG CCA ATG ACA AGA CGC T).
The presence of wild-type gene for Aßb was analyzed by using 3'-(TGA GTT GTG TCT GCA CAC GG) and 5'-(AGT TCA TGG GCG AGT GCT AC). Presence of neomycin gene for mutated Aßb was analyzed by using 3'-(GGA TCG GCC ATT GAA CAA G) and 5'-(CAC CAT GAT ATT CGC CAA GC) oligonucleotide sequences. Presence of both genes indicates the mouse is heterogyzous for the mutated gene.
Immunofluorescence and flow cytometry
Expression of HC of B27 and endogenous H2-Aßb molecules on the cell surface was conducted by immunofluorescence using HC10 (21) (kindly provided by Dr. Hidde Plough, MIT, Cambridge, MA) and 3F12 or AF6.120 (HB163) mAbs (American Type Culture Collection, Rockville, MD), respectively. Briefly, mononuclear cells from peripheral blood were incubated with Abs for 30 min at 4°C. After washing with FACS buffer (PBS containing 1% BSA and 0.05% sodium azide), cells were incubated with a fluorescence-labeled secondary Ab (IgG goat anti-mouse F(ab')2; Accurate Chemical & Scientific Corporation, Westbury, NY). Expression of cell surface molecules was analyzed on 10,000 gated lymphocytes on forward and side scatter by flow cytometry by using CellQuest software.
Peptide synthesis
Three overlapping synthetic peptides covering the third hypervariable region (HV3) of B*2705 molecule were chemically synthesized by solid-phase procedure using Applied Biosystems (Foster City, CA) at Protein Core Facility, Mayo Clinic (Rochester, MN). The amino acid sequences of these overlapping peptides are as follows:B27#1 (57–76): PEYWDRETQICKAKAQTDRE.B27#2 (66–85): ICKAKAQTDREDLRTLLRYY.B27#3 (77–90): DLRTLLRYYNQSEA.
Sequence integrity and homogeneity of these peptides were confirmed by reverse-phase HPLC.
Lymphoproliferative response
A 2-day-long culture in the presence or absence of specific
peptides was conducted to analyze lymphocyte proliferation. Briefly,
mice were immunized with 200 µg of peptide emulsified with CFA
intradermally at the base of the tail (100 µg) and rear footpads (50
µg each). After 7 to 10 days, draining lymph nodes were removed and a
single cell suspension was prepared. RBC were lysed using hypotonic ACK
(ammonium chloride and potassium chloride) solution, and after washing,
mononuclear cells were resuspended in complete media (RPMI 1640 +
10% heat-inactivated FBS + penicillin and streptomycin). Now,
106 cells/well were plated in 96-well flat-bottom
tissue culture plate in the presence or absence of indicated amount of
peptide (Fig. 1A). In some experiments, mAbs (10
µg/ml purified Ab or 20 µl culture supernatant) were added in
addition to the peptide to block specific immune response (Fig. 1B). On the next day, 1 µCi of
[3H]TdR (Amersham, Arlington Heights, IL) was added to
each well, and 24 h later, cells were harvested onto glass fiber
paper. Scintillation fluid was added, and ß emissions were counted
and calculated in cpm. The data were analyzed by using the following
calculations: 
cpm (
cpm) = mean cpm in experimental wells
- mean cpm in control wells without peptide.
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Mice used in the study were between 9 and 12 wk of age and age/sex matched. Experimental groups of mice were i.v. injected with 1 mg of HC10 (human MHC class I HC specific) (21) or 3F12 mAb before transferring them from the barrier facility to the conventional mouse colony. Control groups of mice received either the same amount of an isotype-matched irrelevant Ab or PBS.
Disease monitoring
As described before, mice were monitored for the development of arthritis, nail disease, and other clinical changes (such as skin inflammation and hair loss) twice per week for a period of 12 wk in the conventional area.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B27+ß2mo mice have very
low level expression of MHC class I molecules and few CD8+
T cells. These mice have intact mouse class II molecule
H2-Ab and a normal population of CD4+ T
lymphocytes. To investigate whether the H2-A molecule could be
presenting HLA-B27-derived peptides to CD4+ T cells to
initiate disease in these mice, we first examined the immune response
to peptides derived from HV3 of HLA-B27 molecule. As shown in Figure 1
A,
B27+ß2mo mice showed vigorous
lymphoproliferative response to B27 peptide 57–76 ( cpm >
20,000). These mice also responded to B27 peptide 77–90, but to a
lesser extent. B27+ß2mo mice did
not show lymphoproliferative response to B27 peptide 66–85. To
determine the specificity of the immune response, we added mAbs against
CD4, CD8, and MHC molecules 1 h before addition of B27 peptide
57–76 to the culture. mAbs against the CD4 and H2-A molecules (Fig. 1B) completely inhibited the lymphoproliferative
response. Addition of anti-CD8, anti-B27, and other
anti-mouse MHC class I (H2-Kb and
H2-Db)-specific Abs in lymphocyte cultures with B27 peptide
57–76 did not show such effect on the lymphoproliferative responses
(Fig. 1B). These data suggest that immune response to
B27 peptide 57–76 is mediated by CD4+ T cells and is
restricted to the H2-A molecule. These experiments imply that
B27+ß2mo mice are not tolerized
to self B27 unless these peptide responses represent cryptic
determinants (22). Since H2-A molecule is capable of presenting at
least one of the three B27-derived peptides from HV3 region of the B27
molecule, is it possible that the disease in
B27+ß2mo mice is due to such
presentation of B27-derived peptide by MHC class II molecule. To
directly address this question, we bred MHC class II knockout gene
(Aßo) into
B27+ß2mo mice.
Generation of B27+ß2moAßomice and disease susceptibility
To determine any contribution by endogenous class II molecules in
the disease, MHC class II-deficient, Aßo mice were
mated with HLA-B27 transgenic mice, and the F1 population
was intercrossed to obtain B27+Aßo mice (Fig. 2). B27+Aßo
mice lack expression of MHC class II molecules (H2-A and H2-E), and
thus have a negligible number of CD4+ T cells in the
periphery (Fig. 3). Unlike previously
described B27+ß2mo mice,
B27+Aßo mice do not develop spontaneous
disease (Table I). The
B27+Aßo mice were mated with
ß2moAßo mice to obtain
B27+ß2moAßo mice.
These mice lack normal expression of H2-Kb,
H2-Db, HLA-B27 (data not shown), and H2-A molecules, and
therefore have low levels of mature CD4 and CD8+ T cells in
the periphery.
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B27+ß2moAßo mice
developed arthritis and nail disease within 2 to 3 wk after
transferring them from the specific pathogen-free colony to the
conventional area. Similar to B27-associated spondyloarthropathy in
humans, male mice were primarily affected (Table I). Since these mice
lack functional H2-A and H2-E molecules, it is clear that B27-derived
peptides presented by MHC class II molecules are not the cause of
disease in B27 transgenic animals. Moreover, lymph node cells from
B27+ß2moAßo mice
immunized with B27-derived peptides do not respond to B27-derived
peptides when challenged in vitro (data not shown).
B27+Aßoß2m+/+ mice
did not develop arthritis and nail disease. A few
B27+Aßoß2m+/- mice
showed lower incidence of disease susceptibility (23% among male
animals). The heterozygocity of wild-type ß2m gene
results in lower expression of B27 molecules due to competition with
endogenous class I molecules. A few B27 molecules may reach the cell
surface free/empty. In addition, background genes may also have some
influence in disease susceptibility. In vivo treatment with HC10 Ab decreases incidence of arthritis and nail disease in B27+ß2momice
We have hypothesized previously that the development of
spontaneous disease in
B27+ß2mo mice may be the
result of exogenous Ag presentation by the few ß2m-free
B27 HC expressed on the cell surface. To confirm a direct role of B27
HC, mice were treated with appropriate mAbs to block presentation of
unknown environmental Ags before transferring them from specific
pathogen-free barrier facility. At the same time, control groups of
mice were treated with isotype-matched Ab (L368) or left untreated. As
shown in Figure 4, in vivo treatment with
the HC-specific HC10 Ab decreased the incidence of disease in
B27+ß2mo mice. No such effect was
observed in the group of mice treated with 3F12 mAb. Only 25% of mice
treated with the HC10 Ab developed arthritis and/or nail disease
compared with 60 and 73% of mice in 3F12 (anti-H2-Ab)
or control Ab-treated groups. In vivo treatment with an Ab (ME1)
reactive to [HC of B27+ß2m] complex was
also found ineffective. Since the HC10 Ab recognizes and binds to the
12 peptide binding domains of the HLA-B27
molecule and not the 3 domain (data not shown), in vivo
treatment was thought to block binding and presentation of
environmental Ags by HCs. These results demonstrate that in vivo
treatment with the HC10 Ab blocks presentation of unknown environmental
Ags by HLA-B27 HC, thereby delaying development of spontaneous disease
in B27+ß2mo animals. In addition,
in vivo treatment with HC10 Ab may also interfere with the generation
of B27-derived peptides.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To address the question of whether class II molecules in the B27+ß2mo mice may be presenting a B27-derived peptide, these mice were treated with the 3F12 (anti-H2-Ab) Ab. This treatment had no effect on the disease, suggesting that class II molecules may not have any role in disease pathogenesis. Since B27-ß2mo fullsibs are not susceptible to the disease, we know that class II molecules are not involved in the disease by presenting an exogenous peptide. Since B27+ß2mo mice express low levels of HCs of B27 on cell surface, it is possible that these HCs may be presenting an environmental Ag to initiate the disease. Similarly, presentation of peptide by H2-K and H2-D molecules has been shown in ß2m-deficient mice (24, 25). Our findings on the decreased incidence of spontaneous arthritis and nail changes after treatment with the anti-HC of B27-specific HC10 Ab suggest a role for these B27 HC in the disease process by presenting an unknown environmental peptide.
Since several enterobacteria are known to be part of the normal gut flora in mice, they could be the source of disease-causing peptide in these mice. Although Ags presented in association with MHC-encoded class I molecules are generally derived from intracellular proteins (26), under some circumstances exogenous Ags have been reported to enter class I presentation pathway (27, 28, 29, 30, 31, 32). Processing of bacterial Ag by phagocytic route in mast cells and presentation by the class I molecule to T cells have recently been described (33). Moreover, presentation of exogenous Ags by empty HLA-B27 has been described (34). Furthermore, transfer of inflammatory disease in B27 transgenic rats by bone marrow engraftment suggests the presentation of exogenous Ags by HLA-B27. Very recently, presentation of tissue-associated self Ags in the context of class I via an exogenous processing pathway has been described (35). Our data indicate a direct role for B27 molecules in the disease process.
Even though a role for class II molecules in B27-linked spondyloarthropathies has been minimum, certain observations and hypotheses have raised some questions (12, 13, 19). The first observation is that a low level relative risk in some B27-linked diseases has been attributed to the HLA-DR or HLA-DQ genes (36). The next observation is that class II-restricted CD4+ T cells have been found in affected tissues in certain patients with B27-associated spondyloarthropathies (14, 15, 16). Are these purely bystander lymphocytes or are they generated during chronic infection by an epitope-spreading phenomena, or are they actually effector T cells involved in joint injury? A recent hypothesis has proposed that the disease may be initiated by the presentation of HLA-B27-derived peptides by class II molecules (13). Although B27+ß2mo mice showed immune response to B27-derived peptide (57–76), in vivo treatment with anti-H2-Ab Ab had no effect on the disease. B27+ß2moAßo mice lacking endogenous class II molecules developed spontaneous disease similar to that of B27+ß2mo transgenic mice. Thus, B27-derived peptides presented by class II molecules are not the cause of spontaneous disease in these mice. Prevention of disease with in vivo administration of anti-HC Ab shows that B27 HC play a direct role in the disease pathogenesis. We cannot rule out the possibility that the disease is mediated by the presentation of B27-derived peptides by B27 molecule. Could the slightly reduced incidence in the B27+ß2moAßo mice compared with B27+ß2mo mice be due to reduced levels of CD4+ T cells? We are currently exploring the role of CD4 vs CD8 T cells, the source of peptide and potential immunotherapies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chella S. David, Department of Immunology, Mayo Clinic and Medical School, Rochester, MN 55905. E-mail address:
3 Abbreviations used in this paper: ß2m, ß2-microglobulin; HC, heavy chain; HV3, third hypervariable region.
Received for publication March 20, 1997. Accepted for publication September 18, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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