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The impact of glycosylation on HLADR1restricted T cell recognition of type II collagen in a mouse model.

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ARTHRITIS & RHEUMATISM
Vol. 54, No. 2, February 2006, pp 482–491
DOI 10.1002/art.21565
© 2006, American College of Rheumatology
The Impact of Glycosylation on HLA–DR1–Restricted T Cell
Recognition of Type II Collagen in a Mouse Model
Alexei von Delwig,1 Daniel M. Altmann,2 John D. Isaacs,1 Clifford V. Harding,3
Rikard Holmdahl,4 Norman McKie,1 and John H. Robinson1
Objective. Type II collagen (CII) is a candidate
autoantigen implicated in the pathogenesis of rheumatoid arthritis (RA). Posttranslational glycosylation of
CII could alter intracellular antigen processing, leading
to the development of autoimmune T cell responses. To
address this possibility, we studied the intracellular
processing of CII for presentation of the arthritogenic
glycosylated epitope CII259–273 to CD4 T cells in macrophages from HLA–DR1–transgenic mice.
Methods. HLA–DR1–transgenic mice were generated on a class II major histocompatibility complex–
deficient background, and T cell hybridomas specific for
the glycosylated and nonglycosylated epitope CII259–273
were developed. Subcellular fractionation of macrophages was used to localize CII degradation to particular compartments and to identify the catalytic subtype
of proteinases involved.
Results. We showed that the glycosylated CII259–273
epitope required more extensive processing than did the
nonglycosylated form of the same epitope. Dense fractions containing lysosomes were primarily engaged in
the processing of CII for antigen presentation, since
these compartments contained 1) enzyme activity that
generated antigenic CII fragments bearing the arthritogenic glycosylated epitope, 2) the antigenic CII fragments themselves, 3) CII peptide–receptive HLA–DR1
molecules, and 4) peptide/HLA–DR1 complexes that
could directly activate T cell hybridomas. Degradation
of CII by dense fractions occurred optimally at pH 4.5
and was abrogated by inhibitors of serine and cysteine
proteinases.
Conclusion. Processing of the arthritogenic glycosylated CII259–273 epitope, which is implicated in the
induction of autoimmune arthritis, is more stringently
regulated than is processing of the nonglycosylated form
of the same epitope. Mechanisms of intracellular processing of the glycosylated epitope may constitute novel
therapeutic targets for the treatment of RA.
Inflammation of peripheral joints with destruction and remodeling of the articular cartilage are characteristic features of rheumatoid arthritis (RA). Type II
collagen (CII; ␣1[II]3) forms the backbone of the heteropolymeric fibrils that constitute 80–85% of the total
collagen content of articular cartilage (1). Several lines
of evidence suggest that CII is a candidate autoantigen
in RA. Indeed, immunization of susceptible strains of
mice with CII in adjuvant induces CII-specific T cells
and B cells and results in the development of autoimmune collagen-induced arthritis (CIA) (2,3). Mice
expressing the Aq class II major histocompatibility complex (MHC) molecule (4,5), as well as transgenic mice
expressing human HLA–DRB*0101 and DRB*0401
genes (6–8) (which are associated with RA in humans
[9]), have been shown to be susceptible to CIA. The
strong genetic link between RA and the MHC supports
the role of T cells in the pathogenesis of RA, particularly
during disease initiation and perpetuation (10). CIIspecific T cell responses in RA patients also correlate
with inflammatory activity and radiographic severity in
RA (11), and these responses are directed toward the
immunodominant arthritogenic region CII259–273 (12–14).
It has been proposed that posttranslational modification of self proteins is involved in autoimmunity via
Supported by the Arthritis Research Campaign, UK (grant
MP/R0619).
1
Alexei von Delwig, PhD, DSc, John D. Isaacs, PhD, FRCP,
Norman McKie, BSc, PhD, John H. Robinson, BSc, PhD: University
of Newcastle upon Tyne, Newcastle upon Tyne, UK; 2Daniel M.
Altmann, BSc, PhD: Imperial College School of Medicine, Hammersmith Hospital, London, UK; 3Clifford V. Harding, MD, PhD: Case
Western Reserve University, Cleveland, Ohio; 4Rikard Holmdahl,
MD, PhD: Lund University, Lund, Sweden.
Address correspondence and reprint requests to Alexei von
Delwig, PhD, DSc, Musculoskeletal Research Group, Clinical Medical
Sciences, University of Newcastle upon Tyne, Framlington Place,
Newcastle upon Tyne NE2 4HH, UK. E-mail: alexei.delwig@ncl.ac.uk.
Submitted for publication April 8, 2005; accepted in revised
form October 20, 2005.
482
ANTIGEN PROCESSING OF HUMAN TYPE II COLLAGEN IN A MOUSE MODEL
changes in T cell and antibody specificity or changes in
intracellular antigen processing (15). Posttranslational
glycosylation of CII, in particular O-linked glycosylation
of the K264 and K270 residues located within the arthritogenic CII259–273 region has been shown to correlate
with the enhanced arthritogenicity of CII and the creation of new CII epitopes recognized by autoimmune T
cells in mice and in humans with RA (12,16,17). The
K264 residue has been reported to bind T cell receptors
of autoreactive T cells or to contribute to the binding
affinity of CII259–273 for Aq, HLA–DR1, and HLA–DR4
molecules (18), such that glycosylation of this amino acid
has dramatic effects on class II MHC binding, T cell
recognition, arthritis severity, and production of CIIspecific antibodies (19).
In order for CII-specific T cells to become activated, proteolytic processing of CII by professional
antigen-presenting cells (APCs) is required (20,21). Previous studies have focused on the presentation of undefined CII epitopes (20,22) as well as the CII259–273
epitope (minimal epitope CII260–270) (23). In particular,
it has been shown that the glycosyl side chain of K264
could not be removed by APCs (23,24). However, little
information is available on the mechanisms of proteolytic CII processing for presentation of the glycosylated
epitope CII259–273, which could be the key to understanding the activation of autoimmune T cells during RA.
In this study, we evaluated the intracellular processing of CII in macrophages from HLA–DR1–
transgenic mice for presentation of the glycosylated
CII259–273 epitope. We found that the glycosylated CII
epitope required more extensive processing than did the
nonglycosylated form of the same epitope for presentation to CD4 T cells.
MATERIALS AND METHODS
Antigens. CII purified from normal human cartilage
was purchased from MD BioSciences (Zurich, Switzerland).
Deglycosylation of CII was performed by treatment with
trifluoromethanesulfonic acid:anisol (2:1) for 3 hours on ice,
followed by 1 hour of incubation at room temperature, as
described elsewhere (16). The glycosylated peptide (GIAGFKGEQGPKGET; where K represents GalHyL264) corresponding to epitope CII259–273 containing GalHyL264 was
synthesized using ␤-D-galactopyranosyl-5-hydroxy-L-lysine, as
described previously (25). The nonglycosylated peptide
pCII259–273 was purchased from GenScript (Piscataway, NJ),
and identity was confirmed by mass spectrometry.
Animals. Previously described HLA–DR1–transgenic
mice (designated C57BL/6J0-0 HLA–DR1) carrying full-length
genomic constructs for HLA–DRA1*0101 and HLA–
DRB1*0101 (26) were crossed for more than 6 generations to
483
C57BL/6 Ab–null mice (27) and genotyped by polymerase
chain reaction for the presence of the HLA transgenes. This
newly derived strain of DR1–transgenic mice lacked both
human CD4 and endogenous mouse class II MHC molecules
and was characterized by 1) a normal distribution of DR1⫹
lymphoid cell types in the spleen, 2) the expression of DR1
molecules on professional APCs, but not on CD4 or CD8 T
cells, 3) a normal distribution of T cell receptor (TCR) V␤
family expression among spleen cells, and 4) the susceptibility
to CIA (Von Delwig A, et al: unpublished observations). The
high magnitude of CII-specific T cell responses in DR1transgenic mice suggested that mouse CD4 did not interfere
with antigen presentation by human DR1 molecules. Control
C57BL/6 mice were purchased from Harlan UK (Blackthorn,
UK). Experiments described herein were performed under the
terms of Animals (Scientific Procedures) Act of 1986 and were
authorized by the Home Secretary, Home Office, UK.
Immunization. CII was solubilized in 0.01M acetic acid
at 2.0 mg/ml and then emulsified with an equal volume of
Freund’s complete adjuvant (CFA) containing 4.0 mg/ml of
Mycobacterium tuberculosis (Chondrex, Redmond, WA). One
hundred micrograms of CII emulsified in CFA was injected
subcutaneously at the base of the tail. Ten days later, the
inguinal lymph nodes were removed for testing CII-specific T
cell responses and developing CII-specific T cell lines, as
described previously (6).
Cells. Culture media ingredients, inhibitors, and enzyme substrates were purchased from Sigma (Poole, UK),
except as stated otherwise. Cells were grown in culture medium (RPMI 1640 medium containing 3 mM L-glutamine, 50
␮M 2-mercaptoethanol, 10% fetal bovine serum, and 30 ␮g/ml
of gentamicin). Twenty-two CII-specific DR1-restricted T cell
hybridomas were developed by polyethylene glycol fusion of
BW5147 (TCR␣–/␤–) cells with CD4 T cell lines from CIIimmunized DR1-transgenic mice. The specificity was confirmed in antigen presentation assays using CII and the
synthetic peptides described above. Twenty-one of 22 T cell
hybridomas recognized the nonglycosylated epitope, and 1 T
cell hybridoma was specific for the glycosylated epitope (data
not shown). T cell hybridomas HCII-9.1 (CD3⫹, ␣ /
␤TCR⫹,V␤14⫹; specific for the nonglycosylated peptide) and
HCII-9.2 (CD3⫹,␣/␤TCR⫹,V␤8.1/8.2⫹; specific for the glycosylated peptide) were selected for the antigen presentation
assays.
Macrophages from femoral bone marrow cells obtained from DR1-transgenic mice were grown for 6 days in
culture medium supplemented with 5% horse serum, 1 mM
sodium pyruvate, 10 mM HEPES, and 7.5% of a supernatant
from the L929 cell line as a source of macrophage colonystimulating factor. The human macrophage cell line THP-1
(HLA–DR1/DR2) (TIB202; American Type Culture Collection [ATCC], Rockville, MD) was grown in 96-well plates
(Greiner, Stonehouse, UK) and was stimulated for 24 hours
with 10 ng/ml of phorbol myristate acetate to promote adherence. In all experiments, APCs were stimulated for 24 hours
with 10 units/ml of recombinant interferon-␥ (R&D Systems,
Abingdon, UK).
Antigen presentation assays. Adherent macrophages
(105/well) in 48-well flat-bottomed plates were pulsed with 20
␮g/ml of CII for 3 hours in the absence or presence of the
enzyme inhibitors pepstatin (0.5 mM) (28), phenanthroline
484
(0.1 mM) (29), phenylmethylsulfonyl fluoride (PMSF; 3.0
mM), 20 mM NH4Cl (30), (2S,3S)-trans-epoxysuccinyl-Lleucylamido-3-methylbutane ethyl ester (E-64d; 10 ␮M) (31),
N␣-p-tosyl-L-lysine chloromethylketone (TLCK; 250 ␮M) (32),
and N-p-tosyl-L-phenylalanine chloromethylketone (TPCK; 2.5
␮M) (29), with either cycloheximide (20 ␮M) to block protein
synthesis (33) or brefeldin A (1.0 ␮g/ml) to disrupt Golgi
transport (34). The optimal doses of inhibitors were established in separate dose-response experiments, as reported
previously (30).
To test antigen presentation of CII digests, 40-␮l
aliquots of each digest were added to fixed THP-1 cells in the
presence of the same doses of the enzyme inhibitors PMSF,
E-64d, pepstatin, and phenanthroline to prevent further degradation (35) and then incubated for 18 hours at 37°C. To
localize peptide/DR1 complexes in subcellular fractions, vesicular compartments present in the Percoll gradient fractions
were added to 96-well plates and tested with T cell hybridomas.
Synthetic peptides (at 1.0 ␮M) were added to macrophages or
aliquots of fractions as a positive control for T cell hybridoma
responsiveness.
Cells were fixed in 1.0% paraformaldehyde for 5
minutes, washed thoroughly to remove the fixative, and T cell
hybridomas HCII-9.1 (specific for the nonglycosylated
epitope) and HCII-9.2 (specific for the glycosylated epitope)
were added (5 ⫻ 104/well) and incubated for 24 hours at 37°C.
To measure antigen-nonspecific responses of T cell hybridomas, a dilution series of rat anti-CD3 antibodies (clone KT3;
a generous gift of Dr. C. G. Brooks, University of Newcastle
upon Tyne, Newcastle upon Tyne, UK) were coated on 96-well
plates, and T cell hybridomas were added as above. The
interleukin-2 content of hybridoma supernatants was measured by bioassay as the proliferative response of the cytotoxic
T cell line CTLL-2 (3 ⫻ 104/well) (TIB214; ATCC) during 24
hours of incubation in the presence of 14.8 kBq of 3Hthymidine (specific activity 307 MBq/mg; Amersham, Little
Chalfont, UK). Cells were harvested onto glass-fiber membranes, and radioactivity was quantitated using a direct Beta
Counter (Matrix 9600; Packard, Meriden, CT).
Subcellular fractionation. Macrophages (15–25 ⫻ 106)
were washed with ice-cold RPMI 1640 followed by phosphate
buffered saline, and homogenized in buffer containing 0.25M
sucrose, 10 mM HEPES, pH 7.4, using a Dounce tissue grinder
(Wheaton, Millville, NJ), to obtain 80–85% cell lysis. In some
experiments, macrophages were first incubated with 200 ␮g/ml
of CII for 0, 1, 3, and 5 hours at 37°C in culture medium. Intact
cells and nuclei were removed by 3 cycles of differential
centrifugation (270g for 2 minutes at 4°C), and supernatants
containing postnuclear membranes were layered on 27% Percoll (Amersham), followed by centrifugation (36,000g for 60
minutes at 4°C) in a Sorvall type A-1256 fixed-angle rotor
(Sorvall, Wilmington, DE). Six fractions (numbered 1–6 from
the top of the gradient) of 1.5 ml were collected manually.
Percoll gradient fractions were each tested for
␤-hexosaminidase activity as a marker for the presence of
lysosomal enzymes (34). The reaction mixture containing 50 ␮l
of sample, 150 ␮l of assay buffer (0.1M MES, 0.2% Triton
X-100, pH 6.5), and 50 ␮l of chromogenic substrate (pnitrophenyl-N-acetyl-␤-D-glucosaminide formulated fresh at
1.37 mg/ml in water) was incubated for 90 minutes at 37°C, and
the absorbance was measured at 405 nm. Percoll gradient
VON DELWIG ET AL
fractions were also tested for alkaline phosphodiesterase I
activity as a marker for the presence of plasma membranes
(36). The fractions (50 ␮l) were mixed with 200 ␮l of chromogenic substrate (2 mM thymidine-5⬘-monophospho-pnitrophenyl ester formulated fresh in 40 mM Na2CO3/
NaHCO3 buffer, pH 10.5, containing 0.1% Triton X-100),
incubated for 20 minutes at 37°C, and absorbance was measured at 405 nm.
Protease activity of Percoll gradient fractions was
determined by measuring the release of amino acids and small
peptides from azocasein. Percoll gradient fractions (100 ␮l)
were mixed with 200 ␮l of 2% azocasein (Sigma) dissolved in
0.1M sodium citrate buffer, pH 5.5, containing 0.1% Triton
X-100. After incubation at 37°C for 24 hours, proteolysis was
stopped by adding 200 ␮l of 20% cold trichloroacetic acid.
After centrifugation at 1,000g for 10 minutes, 200 ␮l of the
supernatant was collected, and azocaseinase activity was measured as the absorbance at 405 nm.
Percoll gradient fractions were mixed at a 1:1 ratio with
2.0 mg/ml of CII in 50 mM MES buffers, pH 4.5, 5.5, and 6.5,
and then incubated for 2.5 or 5 hours at 37°C. In some
experiments, the enzyme inhibitors described above were
added. Digestion was stopped by heat-inactivation at 100°C for
3 minutes. Digests were mixed with sodium dodecyl sulfate
(SDS) nonreducing sampling buffer, analyzed by SDS–
polyacrylamide gel electrophoresis (PAGE) on 10% polyacrylamide gels under nondenaturing conditions, and silver-stained
using a Silver Stain Plus kit (Bio-Rad, Hertfordshire, UK)
according to the manufacturer’s instructions.
RESULTS
Mechanisms of CII presentation to T cells. To
study the mechanisms of processing and presentation of
CII in macrophages from HLA–DR1–transgenic mice, T
cell hybridomas specific for the glycosylated (HCII-9.2)
and nonglycosylated (HCII-9.1) CII259–273 epitope were
developed that had no cross-reactivity between the two
forms of the epitope. Deglycosylation of CII resulted in
the complete loss of presentation to T cell hybridoma
HCII-9.2, with little effect on presentation to the HCII9.1 hybridoma (Figures 1A and B). These findings were
consistent with previous observations (16).
Antigen dose-response titrations showed that T
cell hybridoma HCII-9.2 was of low sensitivity to antigen
(Figure 1A), as compared with the higher sensitivity of
HCII-9.1 (Figure 1B). Both CII-specific T cell hybridomas responded similarly in wells coated with anti-CD3
antibodies (data not shown), suggesting the high and low
sensitivity to antigen was related to TCR affinity. When
time of exposure to CII was evaluated, responses to the
glycosylated epitope were detected after 3 hours (Figure
1C), whereas the nonglycosylated epitope was presented
optimally to T cell hybridoma HCII-9.1 after a shorter
ANTIGEN PROCESSING OF HUMAN TYPE II COLLAGEN IN A MOUSE MODEL
485
Figure 1. Mechanisms of antigen presentation of type II collagen (CII). A and B, The
specificity of T cell hybridomas was tested by pulsing macrophages from HLA–DR1–
transgenic mice with different doses of glycosylated and deglycosylated CII, as well as
glycosylated or nonglycosylated peptides. C and D, The kinetics of antigen presentation was
measured by pulsing macrophages from HLA–DR1–transgenic mice with CII for different
periods of time. Ag ⫽ antigen. E and F, The source of HLA–DR1 molecules used for
presentation of CII epitopes was studied by pulsing macrophages for 3 hours with different
doses of CII in the absence or presence of 20 ␮M cycloheximide or 1 ␮g/ml brefeldin A.
After fixation, plates were assayed with T cell hybridoma HCII-9.2, which is specific for the
glycosylated epitope (A, C, and E), or T cell hybridoma HCII-9.1, which is specific for the
nonglycosylated epitope (B, D, and F). Interleukin-2 production by T cell hybridomas was
assayed by 3H-thymidine incorporation into CTLL-2 cells. Values are the mean ⫾ SD cpm
of triplicates. Results are representative of 3 experiments.
period of 1.5 hours (Figure 1D). In experiments with
synthetic peptides, presentation of the glycosylated peptide was also slower (Figure 1C) compared with the
nonglycosylated peptide (Figure 1D).
The source of class II MHC molecules that
presented the glycosylated and nonglycosylated epitope
was determined by pretreating macrophages with the
protein synthesis inhibitor cycloheximide to deplete
newly synthesized class II MHC (33) or with brefeldin A,
an inhibitor of class II MHC transport between the
endoplasmic reticulum and the Golgi complex (34).
Both inhibitors blocked presentation of the glycosylated
and nonglycosylated CII epitopes (Figures 1E and F),
but not the relevant synthetic peptides (data not shown),
suggesting that both forms of the epitope are presented
by newly synthesized class II MHC molecules.
Presentation of the glycosylated epitope was
blocked by raising the endosomal pH with NH4Cl (Fig-
ure 2A), whereas presentation of the nonglycosylated
epitope was only partly inhibited (Figure 2B), suggesting
a different pH dependency for presentation of the 2
forms of the same epitope. When macrophages were
pulsed with CII in the presence of inhibitors of the major
proteinase families, presentation of the glycosylated CII
epitope was completely blocked by inhibitors of cysteine
proteinases (E-64d), aspartic proteinases (pepstatin),
and metalloproteinases (phenanthroline) (Figure 2A),
whereas only partial inhibition was observed in the
responses to the nonglycosylated epitope (Figure 2B).
Presentation of the epitope in both glycosylated and
nonglycosylated forms was also blocked by inhibitors of
serine proteinases (PMSF, TPCK, and TLCK) (Figures
2A and B). Synthetic peptide presentation was not
affected or was only marginally influenced by the inhibitors used (Figures 2C and D). The data showed different sensitivity to inhibitors of antigen presentation of the
486
Figure 2. Effects of enzyme inhibitors on the intracellular processing
of type II collagen (CII) by macrophages. Macrophages from HLA–
DR1–transgenic mice were pulsed with different doses of A and B, CII,
C, glycosylated synthetic peptide, and D, nonglycosylated synthetic
peptide in the presence or absence of the following enzyme inhibitors:
10 ␮M (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane
ethyl ester (E-64d), 0.1 mM phenanthroline, 3.0 mM phenylmethylsulfonyl fluoride (PMSF), 2.5 ␮M N-p-tosyl-L-phenylalanine chloromethylketone (TPCK), 0.5 mM pepstatin, 250 ␮M N␣-p-tosyl-L-lysine
chloromethylketone (TLCK), or 20 mM NH4Cl. Cells were fixed and
then incubated with T cell hybridoma HCII-9.2 (A and C), which is
specific for the glycosylated epitope, or T cell hybridoma HCII-9.1 (B
and D), which is specific for the nonglycosylated epitope. Interleukin-2
production by T cell hybridomas was assayed by 3H-thymidine incorporation into CTLL-2 cells. Values are the mean ⫾ SD cpm of
triplicates. Results are representative of 3 experiments.
glycosylated epitope as compared with the nonglycosylated epitope.
Subcellular localization of CII antigen processing activity. Macrophages were subjected to subcellular
fractionation by Percoll density-gradient centrifugation,
and subcellular fractions were analyzed for enzyme
activity characteristic of cellular compartments. Plasma
membranes were localized to fraction 2 based on the
activity of the plasma membrane enzyme alkaline phos-
VON DELWIG ET AL
phodiesterase I (36), and activity of the lysosomal enzyme ␤-hexosaminidase (37) was confined to dense
membrane fractions 5–6 (Figure 3A). We also used an
azocasein assay to localize total proteinase activity predominantly to membrane fractions 5–6, which correspond to lysosomes (Figure 3A). In native SDS-PAGE
analysis, the band corresponding to the uncleaved CII
␣1(II) chains had an apparent molecular weight of 150
kd, and deglycosylation of CII resulted in the reduction
of the apparent molecular mass from ⬃150 kd to ⬃130
kd (Figure 3B). We detected no visible band corresponding to 100 kd, which could be expected for the
␣1(II) chains. Hence, the higher molecular mass of the
CII preparation used in this study could be explained in
part by its high glycosylation and in part by the unusual
pattern of high SDS binding, which increases its apparent molecular mass in SDS-PAGE analysis (38).
CII was digested with Percoll gradient fractions
as a source of enzyme activity at pH 4.5, 5.5, and 6.5,
which are characteristic of lysosomes, late endosomes,
and early endosomes, respectively (39). Analysis of the
digests on native SDS-PAGE showed that the band
corresponding to the uncleaved CII ␣1(II) chains (⬃150
kd) was reduced in density, coincident with the appearance of a number of smaller fragments with a molecular
mass between 50 kd and 150 kd, after incubation of CII
with dense membrane fractions 5–6, but not with lighter
membrane fractions (Figures 3C–E). No visible band
corresponding to the nonglycosylated CII (⬃130 kd) was
detected, suggesting that the CII preparation from normal human joints used in this study was almost entirely
glycosylated, which is consistent with previously published results (16,23). SDS-PAGE of fractions alone did
not reveal any bands equivalent to CII or its visible
products (results not shown). Collagenolytic activity of
the lysosome-rich fractions was pH-dependent, being
particularly pronounced at pH 4.5 (Figure 3C).
Digests were added to prefixed THP-1 macrophages and assayed with T cell hybridomas to measure
presentation of the glycosylated and nonglycosylated CII
epitope. Intact CII was not presented by prefixed APCs
for specific T cell hybridomas, indicating a requirement
for uptake and/or intracellular processing for presentation to T cells (Figures 4A and B). Fractions 5–6
processed CII for presentation of both forms of the CII
epitope, while the fractions alone did not stimulate T
cell hybridomas (Figures 4A and B). Glycosylated and
nonglycosylated peptides were added to Percoll gradient
fractions from untreated macrophages and assayed with
T cell hybridomas, which also showed presentation in
fractions 5–6 (Figures 4C and D). Taken together, these
ANTIGEN PROCESSING OF HUMAN TYPE II COLLAGEN IN A MOUSE MODEL
487
CII was digested with membrane fraction 6 at pH
4.5 in the presence of enzyme inhibitors. SDS-PAGE
analysis of the digests showed that CII degradation was
reduced by inhibitors of cysteine proteinases, as well as
by inhibitors of chymotrypsin-like (TPCK) and trypsinlike (TLCK) serine proteinases (Figure 5A). Digestion
was marginally affected by inhibitors of aspartic proteinases and metalloproteinases, suggesting that CII was
preferentially degraded in macrophages by lysosomal
serine and cysteine proteinases. To study productive
processing, digests were added to prefixed THP-1 cells
to show that presentation of both epitopes was blocked
by inhibitors of serine proteinases (Figures 5B and C). A
residual level of presentation of the nonglycosylated CII
epitope was detectable in the presence of inhibitors of
aspartic proteinases, cysteine proteinases, and metalloproteinases, whereas the same inhibitors blocked presentation of the glycosylated epitope (Figures 5B and C).
These results are consistent with the data on intracell-
Figure 3. Characterization of enzyme activity in subcellular fractions
of macrophages. A, Fractions 1–6 from Percoll density gradients were
incubated with the chromogenic substrates thymidine-5⬘monophospho-p-nitrophenyl ester (to measure activity of the plasma
membrane–associated enzyme alkaline phosphodiesterase I),
p-nitrophenyl-N-acetyl-␤-D-glucosaminide (to localize the activity of
the lysosomal marker ␤-hexosaminidase), or azocasein (to measure
total proteolytic activity). Enzyme activity was measured as the
absorbance at 405 nm. Fraction 0 represents 27% Percoll alone.
Values are the mean ⫾ SD. B, Analysis of CII before (lane 1) and after
(lane 2) deglycosylation. Molecular weight standards are shown in the
left lane. Analysis of CII digestion by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis of subcellular fractions 1–6 from
macrophages were incubated for 5 hours with CII at C, pH 4.5, D, pH
5.5, or E, pH 6.5, and the digests were resolved on 10% polyacrylamide
gels. CII was incubated with 27% Percoll as a control; the position of
the uncleaved CII is indicated by the arrowhead. Molecular weight
standards are shown in the left lane.
data show that enzyme activity in fractions enriched for
lysosomes productively processed CII for presentation
of the CII259–273 epitope in both glycosylated and nonglycosylated forms to approximately the same degree.
Figure 4. Antigen presentation of type II collagen (CII) epitopes by
fixed THP-1 cells. Subcellular fractions 1–6 of macrophages were
incubated with CII at pH 4.5 (A and B) or were pulsed with synthetic
peptides (C and D). After incubation, digests were added to prefixed
THP-1 cells and assayed with T cell hybridoma HCII-9.2 (A and C),
which is specific for the glycosylated epitope, or T cell hybridoma
HCII-9.1 (B and D), which is specific for the nonglycosylated epitope.
As controls, Percoll gradient fractions were incubated in the absence of
CII or CII was incubated in the absence of fractions before adding to the
THP-1 cells. Interleukin-2 production by T cell hybridomas was assayed
by 3H-thymidine incorporation into CTLL-2 cells. Values are the mean ⫾
SD cpm of triplicates. Results are representative of 3 experiments.
488
VON DELWIG ET AL
ular processing shown in Figure 2. The differences in
sensitivity of SDS-PAGE and T cell assays to aspartic
proteinase and metalloproteinase inhibitors may reflect
the greater sensitivity of the T cell readout compared
with SDS-PAGE in detecting low doses of CII fragments.
Subcellular localization of peptide/HLA–DR1
complexes. Macrophages were pulsed with soluble CII,
subjected to subcellular fractionation, and fractions were
assayed with T cell hybridomas HCII-9.2 and HCII-9.1
to localize peptide/HLA–DR1 complexes. Complexes of
HLA–DR1 with the glycosylated and nonglycosylated
Figure 6. Subcellular localization of peptide/HLA–DR1 complexes in
macrophages. Macrophages were pulsed with type II collagen for the
indicated times and then subjected to subcellular fractionation. Fractions were assayed with A, T cell hybridoma HCII-9.2, which is specific
for the glycosylated epitope, or B, T cell hybridoma HCII-9.1, which is
specific for the nonglycosylated epitope. Interleukin-2 production by T
cell hybridomas was assayed by 3H-thymidine incorporation into
CTLL-2 cells. Values are the mean ⫾ SD cpm of triplicates. Results
are representative of 3 experiments.
Figure 5. Effects of enzyme inhibitors on type II collagen (CII)
digestion. A, CII was digested by lysosomal fraction 6 at pH 4.5 in the
presence or absence of the following enzyme inhibitors: 0.5 mM
pepstatin, 0.1 mM phenanthroline, 10 ␮M (2S,3S)-trans-epoxysuccinylL-leucylamido-3-methylbutane ethyl ester (E-64d), 2.5 ␮M N-p-tosylL-phenylalanine chloromethylketone (TPCK), 250 ␮M N␣-p-tosyl-Llysine chloromethylketone (TLCK), or 3.0 mM phenylmethylsulfonyl
fluoride (PMSF). The digests were resolved on 10% polyacrylamide
gels. Fraction 6 alone served as a control; the position of the uncleaved
CII is indicated by the arrowhead. Molecular weight standards are
shown in the left lane. Digests obtained in the presence of inhibitors
were also added to fixed THP-1 cells and assayed with B, T cell
hybridoma HCII-9.2, which is specific for the glycosylated epitope, or
C, T cell hybridoma HCII-9.1, which is specific for the nonglycosylated
epitope. Interleukin-2 production by T cell hybridomas was assayed by
3
H-thymidine incorporation into CTLL-2 cells. Values are the mean ⫾
SD cpm of triplicates. Results are representative of 3 experiments.
CII epitope were detected after 3 hours in dense membrane fractions 5–6, which correspond to lysosomes,
with a low level of presentation in fractions 2–3, which
contain plasma membranes (Figures 6A and B). Peptide/
HLA–DR1 complexes were more abundant in fractions
5–6 after 5 hours, suggesting an accumulation of complexes in lysosomes.
DISCUSSION
In this study, we addressed whether posttranslational glycosylation affected intracellular processing of
human CII for presentation of the glycosylated epitope
CII259–273, as compared with the nonglycosylated
epitope, to specific CD4 T cell hybridomas in HLA–
DR1–transgenic mice. Posttranslational glycosylation of
CII259–273 epitope created a novel T cell epitope that was
ANTIGEN PROCESSING OF HUMAN TYPE II COLLAGEN IN A MOUSE MODEL
recognized by T cells from HLA–DR1–transgenic mice.
Immunizations of DR1-transgenic mice with CII yielded
a high recovery of T cell hybridomas specific for the
nonglycosylated CII epitope and a low recovery of T cell
hybridomas specific for the glycosylated CII epitope.
This result could be due to the lower affinity of DR1 for
the glycosylated epitope. Indeed, glycosylation of the CII
epitope has been reported to decrease CII peptide
affinity for Aq molecules (40). The result could also be
due to either a higher frequency of T cells specific for
the nonglycosylated epitope or a greater availability of
the nonglycosylated epitope after antigen processing.
Our experimental system used bone marrow–
derived macrophages, since these APCs were previously
shown to preferentially present CII to T cells (22,24).
We demonstrated that CII required intracellular processing for subsequent presentation of both the glycosylated and nonglycosylated CII epitope to T cells, with
notable differences in processing requirements between
the 2 forms of the same epitope. First, the glycosylated
epitope was presented more slowly than the nonglycosylated epitope, suggesting that glycosylation retarded
the rate of formation of peptide/HLA–DR1 complexes,
which is consistent with the demonstration that glycosylation of the CII epitope decreases CII peptide affinity
for Aq molecules (40). Second, the intracellular processing of CII for presentation of the glycosylated epitope
was more sensitive than the nonglycosylated epitope to
treatment of macrophages with inhibitors of cysteine
proteinases, aspartic proteinases, and metalloproteinases, which is consistent with the interpretation that CII
was differentially processed for presentation of the
glycosylated and nonglycosylated epitopes. In addition,
both forms of the epitope were profoundly sensitive to
inhibitors of serine proteinases, which together with the
results of our previous study (30), suggests a major role
of this class of enzyme in antigen processing.
Overall, the pattern of sensitivity to enzyme
inhibitors suggested that presentation of the glycosylated
epitope required more extensive processing than did
presentation of the nonglycosylated epitope. Our data
therefore support the previous proposal that posttranslational glycosylation of autoantigens confers resistance
to proteolysis, with implications for the development of
autoimmunity (15). The T cell response to both forms of
the epitope was dependent upon intact protein synthesis
and Golgi transport, which are required for delivery of
newly synthesized class II MHC to peptide-loading
compartments of APCs, suggesting engagement of the
so-called classic pathway of class II MHC antigen presentation (39).
489
To localize stages of CII degradation to particular intracellular compartments, macrophages were subjected to subcellular fractionation, and the fractions
were used as a source of enzyme activity to digest CII.
Dense membrane fractions corresponding to lysosomes
digested CII at low pH to generate fragments containing
the glycosylated CII epitope that could be presented by
prefixed APCs. Degradation of CII was blocked by
inhibitors of serine and cysteine proteinases, which is
consistent with previous studies implicating cysteine
proteinases (cathepsins B, K, L, N, and S [41–43]) as
well as serine proteinases (44) in intracellular CII degradation and antigen presentation (20). Inhibitors of
serine proteinases, cysteine proteinases, aspartic proteinases, and metalloproteinases reduced the productive
processing of the glycosylated epitope, and to a lesser
extent, the nonglycosylated epitope, which again, is
consistent with differential processing of CII for presentation of the glycosylated and nonglycosylated CII
epitope.
We studied the subcellular localization of HLA–
DR1 complexed with the glycosylated and nonglycosylated CII epitope after intracellular antigen processing
of CII in macrophages and showed that complexes were
present in lysosomes even after a 5-hour pulse with CII,
which indicates a slow egress of these complexes from
antigen-processing compartments to the plasma membrane. This may be a general mechanism of antigen
presentation that is necessary for the sustained activation of autoimmune T cells, which has been shown to
require prolonged antigen presentation on the surface of
APCs (45). When Percoll gradient fractions from untreated macrophages were pulsed with the glycosylated
and nonglycosylated peptides, the formation of peptide/
HLA–DR1 complexes was also detected in dense membrane fractions corresponding to lysosomes, suggesting
the presence of peptide-receptive HLA–DR1 molecules
in lysosomal compartments.
Differential processing of glycosylated and nonglycosylated CII may reflect crucial differences between
RA patients and normal individuals. It has been reported that CII exists largely in the glycosylated form in
healthy cartilage, whereas RA cartilage is characterized
by the presence of both glycosylated and nonglycosylated forms of CII (23). Those investigators also showed
that APCs have a limited capacity to deglycosylate CII,
suggesting low glycosidase activity, which would favor
the differences we observed resulting from antigen processing of distinct pools of glycosylated and nonglycosylated CII. However, it is also possible that deglycosidases, such as lysosomal ␤-galactosidase, influence the
490
VON DELWIG ET AL
induction of arthritis by limiting the availability of the
glycosylated epitope for presentation to arthritogenic T
cells. In this context, it is interesting that a proportion of
mice transgenic for ␤-galactosidase show increased susceptibility to CIA (46).
Posttranslational glycosylation of CII has been
implicated in the balance between tolerance and autoimmunity in mice and in humans (15), although the
particular role of T cells against the glycosylated and
nonglycosylated CII epitope remains poorly understood.
Immune tolerance has been shown to be more pronounced, although not complete, for the nonglycosylated CII epitope, resulting in the appearance of T cells
against both the glycosylated and the nonglycosylated
CII259–273 epitope in the peripheral blood of RA patients
(12). We hypothesize that the requirements for extensive
processing of the glycosylated arthritogenic epitope
shown herein, the low glycosylation status of collagen at
an early age (47), and the inability of some APCs to
process CII (21) may result in a paucity of glycosylated
peptide/HLA–DR1 complexes in the thymus, the inefficiency of negative selection, and the escape of glycosylated CII–specific T cells into periphery. The accumulation of advanced glycation end products with age (48), as
well as the temperature- and pH-dependent changes in
the structural integrity of collagen (49,50) or the increased collagen turnover could enhance the availability
of CII and/or the accessibility of arthritogenic CII
epitopes to antigen processing and lead to the activation
of CII-specific T cells and the development or progression of autoimmune arthritis.
ACKNOWLEDGMENTS
We thank Dr. C. G. Brooks, Institute of Cell and
Molecular Biosciences, University of Newcastle upon Tyne
(Newcastle upon Tyne, UK) for the anti-CD3 antibodies and
Prof. Jan Kihlberg, Umea University (Umea, Sweden) for
synthesis of glycosylated peptides.
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