Ph.D. Dissertation

Investigation of possible immunological

mechanisms in the pathogenesis of a murine

model of rheumatoid arthritis

Anita Hanyecz, M.D.

University of Pécs, Faculty of Medicine

Department of Immunology and Biotechnology

Mentor: Ferenc Boldizsár, M.D., Ph.D.

Program leader: Péter Németh, M.D., Ph.D.

Leader of the Doctoral School: László Lénárd, M.D., Ph.D., D. Sci.

Pécs, Hungary

2013

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TABLE OF CONTENTS

ABBREVIATIONS 5

SUMMARY 7

I. INTRODUCTION 9

I.1. Components and function of the articular cartilage 9

I.2. Structure and function of the aggrecan molecule 10

I.3. Structure and function of the G3 domain of aggrecan molecule 12

I.4. Immunogenicity of cartilage components 13

I.5. Rheumatoid arthritis and its experimental animal models 15

I.5.1. Proteoglycan-induced arthritis (PGIA) 16

I.5.2. Collagen-induced arthritis (CIA) 18

I.5.3. Adjuvant-induced arthritis (AIA) 19

I.6. Dimethyldioctadecylammonium bromide (DDA) as a potential

adjuvant in PGIA and CIA 20

I.7. The rheumatoid arthritis HLA-DRB1 shared epitope 22

I.8. T cell epitope mapping study of aggrecan 24

II. AIMS OF THE STUDY 26

II.1. Investigation of the role of peptide p135-specific lymphocytes and

T cell hybridomas in the induction of arthritis 26

II.2. Investigation of the adjuvant effect of dimethyldioctadecylammonium

bromide in proteoglycan-induced arthritis and collagen-induced arthritis 27

III. MATERIALS AND METHODS 29

III.1. Isolation of antigenic components from cartilage 29

III.1.1. Crude (total) cartilage extracts 29

III.1.2. High-density cartilage proteoglycan 29

III.1.3. Type II collagen 29

III.2. Synthetic peptides 31

III.3. Animals 32

III.4. Immunization protocols 32

III.4.1. Peptide p135 (hyper)immunization experiments and peptide

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priming of lymphocytes 32

III.4.2. Adjuvant studies: comparison of DDA with CFA 33

III.5. Assessment of arthritis 36

III.6. Cell lines and culture media 36

III.7. Generation of p135-reactive T cell hybridomas 36

III.8. Measurement of antigen-specific cell proliferation, cytokine

secretion and antibody production 37

III.9. Flow cytometric analysis of cell surface markers 39

III.10. MHC restriction of p135-reactive T cell hybridomas 39

III.11. Determination of the core epitope and amino acid residues critical

for antigenicity of peptide p135 40

III.12. Determination of the MHC-binding residues of p135H 40

III.13. Identification of T cell receptors of p135-reactive T cell

hybridomas 41

III.14. Cell isolation and transfer of arthritis 42

III.14.1. Transfer of arthritis using p135H-primed lymphocytes

and p135H-specific T cell hybridomas 42

III.14.2. Transfer of arthritis using spleen cells from PG/CFA or

PG/ DDA immunized mice 43

III.15. Statistical analysis 43

IV. RESULTS 44

IV.1. Investigation of the role of peptide p135-specific lymphocytes

and T cell hybridomas in the induction of arthritis 44

IV.1.1. Induction of arthritis in p135H-immunized BALB/c mice

by a single dose of cartilage proteoglycan 44

IV.1.2. In vitro T cell responses in p135H- and p135M-primed

BALB/c mice 45

IV.1.3. Cell surface markers and cytokine secretion of p135H-

and p135M-primed and in vitro restimulated lymphocytes 46

IV.1.4. Induction of arthritis in SCID mice by p135H peptide-

primed lymphocytes 48

IV.1.5. Histopathologic features of arthritis adoptively transferred

by p135H-specific lymphocytes 50

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IV.1.6. Characteristics of p135-specific T cell hybridomas 51

IV.1.7. Determination of the core sequence of p135H that is

critical for recognition by T cell hybridoma H74/4 54

IV.1.8. Stimulatory activity of single-amino acid-substituted

altered peptides 55

IV.1.9. Localization of the I-Ed-binding residues within p135H 56

IV.2. Investigation of the adjuvant effect of dimethyldioctadecylammonium

bromide in proteoglycan-induced arthritis and collagen-induced arthritis 58

IV.2.1. Incidence and severity of PGIA in arthritis-susceptible

BALB/c and C3H colonies 58

IV.2.2. PGIA susceptibility in inbred murine strains 61

IV.2.3. Arthritogenic effect of immunization with DDA and PGs

isolated from various species 62

IV.2.4. Histopathologic features of the peripheral joints and spine

of PGIA-susceptible mice 63

IV.2.5. Mechanisms of action of DDA as adjuvant 65

IV.2.6. Application of DDA in the induction of collagen-induced

arthritis 68

V. NOVEL FINDINGS 70

V.1. Investigation of the role of peptide p135-specific lymphocytes and

T cell hybridomas in the induction of arthritis 70

V.2. Comparison of the adjuvant effects of dimethyldioctadecyl-

ammonium bromide and Freund’s complete adjuvant in proteoglycan-

induced arthritis and collagen-induced arthritis 71

VI. DISCUSSION 73

VII. REFERENCES 83

VIII. BIBLIOGRAPHY 95

IX. ACKNOWLEDGEMENTS 102

X. APPENDIX 103

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ABBREVIATIONS

ABC chondroitinase ABC

ACPA anti-citrullinated protein/peptide antibody

ADAMTS a disintegrin and metalloproteinase with thrombospondin motifs

AIA adjuvant-induced arthritis

Ala alanine

APC antigen presenting cell

Arg arginine

Asp aspartic acid

BSA bovine serum albumin

CII type II collagen

CD cluster of differentiation

cDNA complementary deoxyribonucleic acid

CFA Freund’s complete adjuvant

CIA collagen-induced arthritis

CLD C-type lectin module

cpm counts per minute

CRT calreticulin

CS chondroitin sulfate

C-terminus carboxyl-terminus

CTLL cytotoxic T lymphocyte line

DC dendritic cell

DDA dimethyldioctadecylammonium bromide

DMEM Dulbecco's modified Eagle's medium

E.coli Escherichia coli

EGF epidermal growth factor(-like)

ELISA enzyme-linked immunosorbent assay

endo-β-gal endo-β-galactosidase

FBS fetal bovine serum

G1, G2, G3 globular domains 1, 2, 3

GAG glucosaminoglycan

Glu glutamic acid

Gly glycine

HA hyaluronic acid

HLA human leukocyte antigen

HSP heat shock protein

ID intradermal

IDO indoleamine 2,3 dioxygenase

IFA Freund’s incomplete adjuvant

IFN interferon

IGD interglobular domain

IgG immunglobulin G

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IL interleukin

IP intraperitoneal

KS keratan sulfate

Leu leucine

LP link protein

LPS lipopolysaccharide

Lys lysine

mAb monoclonal antibody

MHC major histocompatibility complex

MMP matrix metalloproteinase

MTP metatarsophalangeal

MW molecular weight

NA not applicable

NCI National Cancer Institute

ND not detectable

NE not evaluated

NO nitric oxide

N-terminus amino-terminus

OA osteoarthritic

p135B bovine peptide 135

p135D canine peptide 135

p135H human peptide 135

p135M mouse peptide 135

p135P porcine peptide 135

PBS phosphate buffered saline

PG proteoglycan

PGIA proteoglycan-induced arthritis

RA rheumatoid arthritis

RNA ribonucleic acid

SC subcutaneous

SCID severe combined immunodeficiency

SCR module complement regulatory protein-like module

SD standard deviation

SE shared epitope

SI stimulation index

TCR T cell receptor

Th cell T helper cell

TNF tumor necrosis factor

Treg regulatory T cell

Tyr tyrosine

WT wild type

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SUMMARY

Rheumatoid arthritis (RA) is a systemic autoimmune disease and its targeting of the

joints indicates the presence of a candidate autoantigen(s) in synovial joints [1-3].

Patients with RA show cellular immune responses in their peripheral blood to cartilage

matrix components such as proteoglycan aggrecan [1, 4]. The most evident source of

these autoantigens is the immunoprivileged articular cartilage, which is avascular, and is

therefore not subject to immunologic surveillance. One of the most relevant animal

models of rheumatoid arthritis appears to be proteoglycan (aggrecan)-induced arthritis

[3, 5]. Although the underlying mechanisms are still unclear, T cells, particularly CD4+

lymphocytes, seem to play a crucial role in the initiation of the disease [6-9]. Earlier

studies explored numerous T cell epitopes along the core protein of human aggrecan

[10, 11], and one of the epitopes (called peptide p135H) present in the G3 domain at the

C-terminus of the human aggrecan appeared to be involved in arthritis induction.

In the first part of the study, we made an effort to study the immunologic function and

determine the fine epitope structure of this synthetic peptide p135H

((2373)TTYKRRLQKRSSRHP), which contains a highly homologous sequence motif

of the “shared epitope” (QKRAA), a sequence that is overrepresented in numerous RA-

associated HLA-DR4 alleles [12] and common human pathogens [13-18]. To further

investigate whether the T cell epitope represented by p135H might be directly involved

in the pathogenesis of the disease, we transferred p135H-stimulated lymphocytes from

p135H-primed BALB/c mice into SCID mice. Despite differences in the amino acid

sequences between the human and mouse peptide and the cryptic character of the

epitope, we were able to show that the transfer of p135H-reactive lymphocytes into

“presensitized” SCID mice leads to a rapid development of arthritis. Since the G3

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domain of cartilage proteoglycan aggrecan with the p135 sequence is “lost” during the

normal metabolic turnover of cartilage proteoglycan or in pathologic conditions [19], an

antigenoriented T cell migration into joints of presensitized (susceptible) individuals

may contribute to the organ-specificity of RA.

In the second part of the study, we compared the effects of

dimethyldioctadecylammonium bromide (DDA), a potent nonirritant adjuvant with

those of Freund’s complete adjuvant (CFA) in arthritis-susceptible murine strains. As a

result, the overall immune responses (antibody production and antigen-specific T cell

responses) were highly comparable when human proteoglycans (PG) in CFA or in DDA

were used, however the use of DDA accelerated the development of a more severe

arthritis, and suboptimum doses of PG or type II collagen antigens were able to induce

inflammation. We showed that DDA exerts a strong stimulatory effect via the activation

of nonspecific (innate) immunity and forces the immune regulation toward Th1

dominance. Since a number of potential autoantigens (e.g. type II collagen,

proteoglycan aggrecan) can be identified in various subsets of RA patients, it may be an

attractive hypothesis that seemingly innocuous compounds may exert an adjuvant effect

in humans and may create the pathophysiologic basis of autoimmunity in susceptible

individuals via the activation/stimulation of innate immunity.

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I. INTRODUCTION

I.1. Components and function of the articular cartilage

Depending on the composition of the matrix, cartilage in human body is classified into

elastic, fibro-cartilage, fibro-elastic and hyaline cartilage. Gliding surfaces of synovial

joint are covered with a specialized type of hyaline cartilage, called “articular cartilage”.

Hyaline cartilage provides a low-friction gliding surface, minimizes peak pressures on

the subchondral bone with increased compressive strength and is known to be wear-

resistant under normal circumstances. Hyaline articular cartilage is aneural, avascular

and alymphatic structure [20].

Chondrocytes form only 1–5% volume of the articular cartilage and are sparsely spread

within the matrix. Chondrocytes synthetize all the matrix components, regulate matrix

metabolism and organize the collagen, proteoglycans and non-collagenous proteins into

a unique and highly specialized tissue. Chondrocytes receive their nutrition by diffusion

through the matrix [20].

Sixty-five to eighty per cent of wet weight of the cartilage is formed by water. It allows

load-dependent deformation of the cartilage, provides nutrition and medium for

lubrication, creating a low-friction gliding surface [20].

Collagen forms 10–20% of wet weight of the articular cartilage. Type II collagen forms

the principal component (90–95%) of the macrofibrilar framework and provides a

tensile strength to the articular cartilage [20].

Proteoglycans (PG) are protein polysaccharide molecules, form 10–20% wet weight and

provide a compressive strength to the articular cartilage. There are two major classes of

PGs found in articular cartilage, large aggregating PG monomers or aggrecans and

small PGs including decorin, biglycan and fibromodulin. They are produced inside

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the chondrocytes and secreted in the matrix, and their function is to maintain the fluid

and electrolyte balance in the articular cartilage [20].

I.2. Structure and function of the aggrecan molecule

Aggrecan is a modular proteoglycan with multiple functional domains. Its core protein

consists of three globular regions, termed G1, G2 and G3, each containing cysteine

residues that participate in disulphide bond formation. The G1 and G2 regions are

separated by a short interglobular domain (IGD), and the G2 and G3 regions are

separated by a long glycosaminoglycan (GAG)- attachment region, which consists of

adjacent domains rich in keratan sulphate (KS) and chondroitin sulphate (CS) (Figure

1.) [21].

Figure 1. Schematic representation of the aggrecan structure

(HA: hyaluronic acid, CS 1, CS 2: chondroitin sulfate domains 1 and 2; KS: keratan sulfate; G1, G2, G3:

globular domains; LP: link protein). Figure is adapted from “Effects of Glucosamine and Chondroitin

Sulfate on Cartilage Metabolism in OA: Outlook on Other Nutrient Partners Especially Omega-3 Fatty

Acids” by Jerosch J, Int J Rheumatol 2011;2011:969012.

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The G1 region is at the amino-terminus of the core protein, and can be further sub-

divided into three functional domains, termed A, B1 and B2, with the B-type domains

being responsible for the interaction with hyaluronic acid (HA) [21].

The G2 region also possesses two B-type domains, but does not appear to interact with

HA, and at present its function is unknown [21].

The G3 region resides at the carboxyl-terminus of the core protein and contains a

variety of distinct structural domains [21]. This domain has homology with the C-type

lectin but to date no distinct carbohydrate binding has been identified. It has been shown

that aggrecan via this domain can interact with certain matrix proteins containing EGF

(epidermal growth factor)-repeats. These include the fibrillins, the fibulins as well as

tenascins. These molecules in themselves can form complex networks. Therefore,

aggrecan can be complexed in the aggregates via its N-terminal domain on one hand

and interact with other assemblies of macromolecules via its C-terminal domain. The

G3 domain is also essential for normal posttranslational processing of the aggrecan core

protein and subsequent aggrecan secretion [22].

Aggrecan molecules do not exist in isolation within the extracellular matrix, but as PG

aggregates. Each aggregate is composed of a central filament of HA with up to 200

aggrecan molecules radiating from it, each interaction to be stabilized by the presence

of a link protein (LP) (Figure 1.). PG aggregate structure is influenced by three

parameters – the length of the HA, the proportion of LP, and the degree of aggrecan

processing. Two molecular forms have been described for PG aggregates extracted

directly from cartilage without the use of dissociative agents. These forms appear to

differ in their LP content, and may have different functional characteristics. It is the

large size of the PG aggregates and their entrapment by the collagen framework of the

tissue that results in aggrecan retention in the extracellular matrix [21].

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Aggrecan molecules rarely exist in an intact form in the PG aggregates of the cartilage

matrix, but instead are subject to extracellular proteolytic processing of their core

proteins. In arthritic diseases, cartilage undergoes irreversible destruction in response to

various catabolic stimuli. Under such conditions, aggrecan molecules are known to be

rapidly degraded and released from the cartilage matrix, followed by the degradation of

the matrix collagens. The primary cause of aggrecan degradation is attributed to

proteolysis by members of the matrix metalloproteinase (MMP) and disintegrin and

metalloproteinase with thrombospondin motifs (ADAMTS) families of enzymes. These

proteinases can cleave aggrecan at specific sites within a proteinase-sensitive region of

the aggrecan core protein located within the IGD. Cleavage at this site results in the loss

of the part of the aggrecan molecule bearing the KS- and CS-attachment domains, while

the G1 domain remains attached to the HA filament and LP in the tissue. Moreover, five

additional cleavages attributed to aggrecanases, have been described within the CS-2

region. Cleavage at these sites contributes to the C-terminal truncation of aggrecan, the

loss of the GAG chains, and the loss of tissue function [23, 24].

I.3. Structure and function of the G3 domain of aggrecan molecule

The G3 region resides at the carboxyl-terminus of the core protein of aggrecan, and is

structurally distinct from the G1 and G2 domains. It contains an epidermal growth

factor-like module (EGF1), a calcium-binding EGF module (EGF2), a C-type lectin

module (CLD), and a complement regulatory protein-like module (SCR). The CLD is

constitutively expressed and mediates binding to other extracellular matrix molecules,

e.g. tenascin-R, fibulin-1, fibulin-2 and fibrillin-1 [22]. Other modules of the G3 domain

are subject to alternative splicing. The expression of the SCR module is variable, but in

humans this module is usually present, regardless of age. The EGF1 and EGF2

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modules are expressed to a lower extent (25–28 % and 5–8%, respectively, in humans)

[25, 26]. The EGF2 module is highly conserved and uniformly expressed at low levels

in several different species, whereas the less conserved EGF1 module is expressed to

different degrees in various species. These differences and the alternative splicing of the

EGF-like repeats may reflect different functions for the EGF1 and EGF2 modules in

different species. Expression of the EGF modules could constitute a mechanism for

feedback regulation of differentiation and proliferation of chondrocytes. In addition,

alternative splicing of modules in the G3 domain could affect aggrecan GAG

substitution and transport through the secretory pathway. The alternative splicing of the

flanking EGF and SCR modules could also have a regulatory function by modulating

the C-type lectin-mediated interactions [22].

Loss of aggrecan is a major feature of cartilage degradation associated with arthritis

[27]. There is an age-related loss of the G3 domain of aggrecan, and 92% of the G3

domain is lost as part of the normal turnover of the PGs, whereas the rest of the

molecule, which is bound to HA, is retained in the cartilage [19].

I.4. Immunogenicity of cartilage components

Cartilage is one of the few immunologically privileged tissues in the body in that it is

essentially avascular and therefore not subjected to close “internal” immunological

surveillance. When it gets degraded, however, uniquely antigenic molecules become

exposed, released and subsequently recognized by the immune system. Thus, articular

components may trigger and maintain immune responses to these antigens [28, 29].

Several autoantigens are described in RA, including a variety of proteins that become

citrullinated in diseased joints. Citrullination is a physiological process of arginine

deimination that occurs during apoptosis and inflammation. This process results in

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modification of arginine-containing proteins, which can give rise to sets of neo-self-

antigens in individuals bearing at-risk human leukocyte antigen (HLA) alleles [30].

Approximately 70% of RA patient sera contain anti-citrullinated protein/peptide

autoantibodies (ACPA) [31]. This reactivity reflects autoantibody production towards a

group of citrullinated autoantigens modified post-translationally, including fibrinogen,

vimentin, type II collagen (CII) and enolase [32]. ACPAs develop up to 15 years prior

to the onset of RA, with increasing titres and peptide reactivities as disease onset

becomes imminent [33]. Citrullinated proteins have been demonstrated in inflamed

tissues in RA, and ACPAs are induced in a number of mouse models of inflammatory

arthritis [34-36]. Although citrullination is ubiquitous in response to stress and

inflammation, ACPAs are highly specific for RA and are associated with more severe

joint damage and radiographic outcome [31, 37, 38]. Moreover, several recent papers

have shown convincing cytokine responses made by RA patient T cells in response to

citrullinated aggrecan and vimentin epitopes [39, 40].

The immune attack on the joints could also be initiated by a cross-reactive immune

reaction in response to unrelated antigens by the mechanism of “molecular mimicry”.

The net result of such autoimmune reactions could be further destruction of cartilage

and release of more autoantigens. This could lead to a chronic, self-perpetuating

inflammation in genetically predisposed individuals who are prone to develop these

autoimmune reactions [41]. Although autoimmunity to cartilage PGs has been studied

less intensively than autoimmunity to CII, cartilage PG (aggrecan) is considered as

causal/contributing factor in rheumatoid joint disease [42].

Patients with RA show cellular immune responses in their peripheral blood to PG [1, 4].

The incidence of immune response to PG in the patients varied from 5 to 85%,

depending upon the study. Cellular immunity to PG has also been described in

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patients with juvenile rheumatoid arthritis [4]. Furthermore, immunoreactive fragments

of PG have been demonstrated in the synovial fluids of patients with RA [43]. These

facts all suggest that PG aggrecan may play an important role in the development of

autoimmunity against peripheral joints.

I.5. Rheumatoid arthritis and its experimental animal models

Rheumatoid arthritis is one of the most frequent systemic autoimmune diseases, it

affects approximately 1% of the human population with a female preponderance.

Genetic factors play a significant role in RA susceptibility. However, the concordance

of this disease in monozygotic twins is only 15%. It has been therefore hypothesized

that while RA susceptibility is determined genetically, disease onset may depend on

non-genetic (i.e. environmental), epigenetic or posttranslational events [44].

Several lines of evidence indicate that the effector mechanism, which initially attacks

small joints, is T cell driven [45, 46]. As a result, an aggressive synovial pannus

develops, which destroys articular cartilage and bone, leading to massive ankylosis and

deformities of peripheral joints. The disease has a progressive character, with the

involvement of more and more joints. Although RA is a systemic autoimmune disease,

its targeting of the joints indicates the presence of a candidate autoantigen(s) in synovial

joints [1-3]. There are numerous rodent models that simulate some or many of the

clinical, immunological or histopathological features of the disease. It has become a

strong working hypothesis that major histocompatibility complex (MHC) and non-MHC

genetic components share loci that are common in various autoimmune diseases, and in

corresponding animal models. The most relevant animal models of rheumatoid arthritis

appear to be those induced by cartilage matrix components such as proteoglycan

aggrecan or type II collagen [5, 6, 47]. The pathologic basis in both model systems

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appears to be cross-reactive immune reactions, that is, the T cells and antibodies raised

against the immunizing heterogeneous (human, bovine) cartilage antigens recognize and

subsequently attack the mouse’s own tissues (self) [10, 48-52].

I.5.1. Proteoglycan-induced arthritis (PGIA)

Systemic immunization of genetically susceptible strains of mice with human cartilage

proteoglycan that has been depleted of glycosaminoglycan side chains leads to the

development of progressive polyarthritis [3, 5]. PGIA shows many similarities to RA in

humans, as indicated by findings of clinical assessments, radiographic analyses,

scintigraphic bone scans, laboratory tests and histopathologic studies of diarthrodial

joints [3, 5, 49, 53].

PGIA was first described in the BALB/c strain [5], but certain C3H colonies (e.g.

C3H/HeJCr) were also found to be susceptible to PGIA [53]. During the immunization

protocol female mice are injected intraperitoneally (IP) with 100 μg of cartilage PG

(measured as protein) in Freund’s complete adjuvant (CFA), followed by injection of

the same doses of PG in Freund’s incomplete adjuvant (IFA), on days 21 and 42. The

initial clinical manifestations of joint inflammation (swelling and redness) appear after

the third to fourth IP injection of antigen, depending on the source of PG antigen,

adjuvant and BALB/c or C3H colony used [5, 10, 48, 49, 53]. Joint inflammation starts

as polyarticular synovitis in small peripheral joints. During the early phase,

lymphocytes and polymorphonuclear leukocytes invade the synovium. This is followed

by gross, “tumorlike” proliferation of synovial lining cells and fibroblasts. Once an

animal develops arthritis, repeated “spontaneous” episodes of inflammation result in

complete destruction of the articular cartilage and erosion of the subchondral bone,

which leads to severe deformities of the peripheral joints [5, 48, 49, 53].

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The disease is polygenic, has a recessive inheritance, and MHC and non-MHC

components are critical for the development of arthritis [54-57].

PGIA in BALB/c mice is a T cell-dependent and (auto)antibody/B cell-driven disease.

Antibodies against immunizing (human) PG appear during the second/third week of

immunization. T cell response to PG is detectable approximately 5-7 weeks after the

start of immunization, and along the course of the disease the humoral and cellular

immune responses slowly decline as the disease becomes chronic and less active [5, 48,

49, 53].

CD4+ T cells have been implicated in the development of PGIA by observations that

anti-CD4 monoclonal antibody (mAb) treatment prevents arthritis, and that the transfer

of disease requires T cells from arthritic animals [7, 58, 59]. BALB/c mice are

genetically predisposed to a T helper (Th)2-type immune response, however,

immunization of these mice with PG induces a higher ratio of interferon (IFN)-γ to

interleukin (IL)-4, indicating that PGIA is a Th1-type response, and immunization with

PG in CFA is a sufficient Th1 stimulus to overcome the genetic inclination toward

development of a Th2-type response [60].

Treatment with Th2 cytokines before the onset of arthritis prevents the development of

arthritis in IL-4-treated animals and, to a lesser extent, in IL-10-treated animals,

indicating that a switch from a Th1-type response to a Th2-type response is critical for

the control of joint inflammation associated with arthritis [60].

In PGIA, neutralization of IFN-γ inhibits arthritis and IFN-γ−/−

mice develop arthritis

with delayed onset and reduced severity in comparison with wild-type (WT) mice.

These findings indicate that IFN-γ is an important pro-inflammatory cytokine

promoting disease severity in PGIA [60, 61].

IL-17 has emerged as an important proinflammatory T cell cytokine in several models

18

of arthritis [62-64]. Contrary to a dependence on IL-17 in other models of arthritis (e.g.

collagen-induced arthritis), in PGIA, IL-17–deficient mice develop arthritis similar to

WT, demonstrating that a robust IFN-γ response in WT mice readily suppresses the IL-

17 response, making the contribution of IL-17 to inflammation negligible [65].

I.5.2. Collagen-induced arthritis (CIA)

Collagen-induced arthritis was first elicited in rats following a single intradermal

injection of type II collagen (CII) emulsified in Freund's adjuvant [6]. Further studies

demonstrated that a similar pathology could also be induced in primates [66] and in

susceptible strains of mice [47]. CIA can be induced using native autologous or

heterologous CII and is specific to CII, since immunization with types I or III collagen

fail to induce disease [6, 47]. While either IFA or CFA can be used to trigger CIA in

rats, the induction of disease in mice generally requires the presence of heat-killed

Mycobacterium tuberculosis in CFA [47]. Immunization with CII/CFA results in a rapid

and severe polyarthritis of the peripheral articular joints that first appears around 3–4

weeks after antigen challenge and becomes progressively worse for approximately 2–4

weeks before slowly waning. Whilst the pathology is similar when CIA is induced with

either autologous or heterologous CII, the nature of the disease differs; autologous CII

induces a more chronic disease with a delayed onset and reduced penetrance [67, 68]. In

both cases the histopathology of inflammatory arthritis resembles human RA. Like RA,

CIA is characterized by the presence of fibrin deposition, hyperplasia of synovial cells,

periosteal bone formation, mononuclear infiltrates, pannus formation and eventual

ankylosis of one or more articular joints [6]. While the precise mechanisms by which

immunization with heterologous or autologous CII in CFA leads to a chronic arthritis in

susceptible mice are not known, there are considerable data to implicate CII-reactive

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CD4+ T cells as the primary mediators of disease induction, and complement-fixing

anti-CII autoantibody production by B cells as the major immune mechanism leading to

the localized chronic inflammatory response [69]. CIA is classified as a Th1-mediated

disease based on the abundant IFN-γ production [70]. However, the role of IFN-γ in

CIA is more complex. Complete elimination of IFN-γ or IFN-γ receptor signaling leads

to exacerbation of disease [71, 72]. On the other hand, neutralization of IFN-γ at an

early stage of disease inhibits arthritis [73]. The ability of IFN-γ to suppress Th17 cells

appears to account for augmented disease in IFN-γ−/−

or IFN-γ receptor deficient mice

in CIA, as inhibition of IL-17 with neutralizing antibodies is able to suppress arthritis

[74]. Autoreactivity to cartilage CII in human RA patients, although not a defining

feature of the disease, has been clearly demonstrated [75, 76].

I.5.3. Adjuvant-induced arthritis (AIA)

Adjuvant-induced arthritis can be induced in rats by a single injection of Freund's

adjuvant, containing Mycobacterium tuberculosis [77]. Arthritis develops at around 10–

45 days after injection and generally subsides after a month. The chief pathological

features of adjuvant arthritis include oedema, mononuclear and polymorphonuclear cell

infiltrations, pannus formation, periostitis, and erosion of cartilage and bone. Although a

link between immunity to 65-kD heat shock proteins and the induction of AIA is

suspected [78], no single mycobacterial immunogen has been identified which is

responsible for the arthritogenic response in this model. Rather, the induction of AIA

has been attributed to a mycobacterial cell wall component, muramyl dipeptide, which

is immunostimulatory but does not evoke a specific immune response [79]. In addition,

a number of adjuvants which lack immunogenic properties have been shown to induce

arthritis in susceptible strains of rats, including avridine [80] and Freund's incomplete

20

adjuvant [81]. Arthritis, bearing many similarities to RA, is also observed in mice

following administration of pristane [82]. Doubts as to the immunological nature of

these diseases were dispelled by the findings that anti-T cell treatments prevent the

induction of arthritis [81, 83], susceptibility is influenced by genes within the MHC

locus [84], and arthritis can be adoptively transferred by T cells [85, 86]. Neutrophils

are also key players in the pathogenesis of AIA, since antineutrophil antibody treatment

was shown to inhibit the development of arthritis [87]. Regarding to cytokines and

chemokines, the production of tumor necrosis factor (TNF)-α, IL-1β, macrophage

inflammatory protein 1α and epithelial neutrophil–activating peptide 78–like protein

was shown to be abundant prior to and during the course of AIA, while that of IL-6 and

JE, the murine homolog of monocyte chemoattractant protein 1, was elevated in the late

phase of AIA [88].

The mechanism of arthritis induction following the administration of adjuvants is

unknown, but one possibility is that there is an increase in the activity of antigen-

presenting cells (APC) [89], leading to the presentation to autoreactive T cells of a

hitherto unrecognized or “sequestered” endogenous antigen. The possibility that human

RA could also be triggered by exposure to environmental factors with adjuvant-like

activity has been highlighted by recent studies in which it was found that arthritis could

be induced in DA rats by percutaneous exposure to adjuvant oils [90] or even a mineral

oil-containing cosmetic product [86, 91].

I.6. Dimethyldioctadecylammonium bromide (DDA) as a potential adjuvant in

PGIA and CIA

A number of animal models can simulate human RA, but all require antigen challenge

with adjuvant [92, 93]. Mycobacterial heat-shock proteins (HSP) present in CFA are

21

known to be very potent nonspecific immunostimulators, and these proteins

significantly contribute to, and potentiate, the systemic T cell response [77, 94]. In

subsequent injections, the antigen can be administered in emulsion with mineral oils

(IFA) that do not contain mycobacterial components. However, these bacterial HSPs of

CFA cause numerous undesired side effects, including CFA-induced irritation and

granuloma formation with subsequent adhesions in the peritoneal cavity, and immune

reactions to mycobacterial components [93].

Dimethyldioctadecylammonium bromide (DDA) belongs to the group of lipophilic

quaternary amines that were described more than 35 years ago as a potential adjuvant

[95]. DDA as an adjuvant has been used successfully and without side effects in

vaccines administered to children and pregnant women [96, 97]. It is also widely used

as a detergent in cosmetic compounds and fabric softeners [98]. It is a positively

charged compound (MW 631 daltons) with a monovalent counter-ion (bromide), and 2

long alkyl chains that are hydrophobic [95, 98]. Because of the lipophilic chains, DDA

is poorly soluble in water but forms a semicolloidal polycationic liposomal micelle

structure at a temperature of 40°C or higher [98, 99]. DDA is a highly potent

immunostimulator, especially with negatively charged antigens, provoking strong

delayed-type hypersensitivity [98, 100-102]. It is a powerful, nonirritant adjuvant and,

via T cell stimulation, significantly enhances antigen-specific B cell activation and

immunoglobulin production [103, 104]. A special benefit of the use of DDA in

numerous rodent models of autoimmunity is that this adjuvant forces the

immunoregulation toward Th1 type response [105-107]. DDA can enhance the adjuvant

effect of other adjuvants [92] and even potentiate the arthritogenic effect of IFA [92] or

CFA [108] in oil adjuvant–susceptible strains of rats. However, DDA as an adjuvant has

never previously been tested in PGIA or CIA.

22

I.7. The rheumatoid arthritis HLA-DRB1 “shared epitope”

Altough the pathogenesis of RA is not fully understood, previous studies have shown

that RA is closely associated with HLA-DRB1 alleles that code a five amino acid

sequence motif in residues 70–74 of the DRβ chain [12]– commonly referred to as the

“shared epitope” (SE). The better-known SE-coding alleles include members of the

HLA-DRB1*04 allele group, HLA-DRB1*0101 or *0102, HLA-DRB1*1402 and HLA-

DRB1*1001 [44]. The disease in SE-positive patients begins earlier and is more erosive

than in SE-negative individuals [109]. The mechanism underlying the effect of SE in

RA is unclear. Based on the known role of MHC molecules in antigen presentation, the

prevailing hypotheses postulate that either selective binding and presentation of

arthritogenic self-peptides [110], molecular mimicry with foreign antigens [14, 17] or T

cell repertoire selection [111] could be involved [112]. Notwithstanding their

plausibility, these hypotheses are difficult to reconcile with the fact that data supporting

antigen-specific responses as the primary event in RA are inconclusive [113].

Additionally, several other human diseases have also been shown to be associated with

SE-encoding DRB1 alleles, including polymyalgia rheumatica [114], giant cell arteritis

[114], type I diabetes [115], erosive bone changes in psoriatic arthritis [116],

autoimmune hepatitis [117] and early-onset chronic lymphoid leukemia [118].

Furthermore, the increasing incidence of RA with age and the positive correlations

observed between the number of copies of SE alleles and RA severity and disease

penetrance are all inconsistent with an antigen presentation-based mechanism [113].

It has been recently demonstrated that SE acts as a potent immune-stimulatory ligand

that activates a nitric oxide (NO)-mediated pathway in other cells and can polarize T

cell differentiation toward Th17 cells, a T cell subset that has been implicated in the

pathogenesis of autoimmune diseases, including RA. Dendritic cells (DC) are

23

strategically positioned in the interface between the innate and adaptive immune

systems. In addition to their antigen presentation role, DCs also induce tolerance

through cross-talk with regulatory T (Treg) cells. In CD11c+CD8

+ subset of murine DCs

the SE inhibited the enzymatic activity of indoleamine 2,3 dioxygenase (IDO), a key

enzyme in immune tolerance and T cell regulation, while in CD11c+CD8

− DCs the

ligand activated robust production of IL-6. When SE-activated DCs were co-cultured

with CD4+ T cells, the differentiation of Foxp3

+ Treg cells was suppressed, while Th17

cells were expanded. The polarizing effects could be seen with SE-positive synthetic

peptides, but even more so, when the SE was in its natural tri-dimensional conformation

as part of HLA-DR tetrameric proteins. In vivo administration of the SE ligand resulted

in higher abundance of Th17 cells in the draining lymph nodes and increased production

of pro-arthrogenic cytokine IL-17 by splenocytes (Figure 2.)[112].

Figure 2. A proposed model of the role of the “shared epitope” (SE) ligand in autoimmunity

The effect of the SE ligand is the end result of its interaction with two dendritic cell (DC) subsets. The SE

ligand, expressed on MHC class II-positive cells, interacts with cell surface calreticulin (CRT) (green)

with resultant nitric oxide (NO) production. In CD8+ DCs, increased NO levels inhibit the enzymatic

activity of indoleamine 2,3 dioxygenase (IDO), which leads to decrease tryptophan metabolites and

reduced generation of regulatory T cells (Tregs). In CD8− DCs, the SE ligand activates production of IL-6

and, in the presence of lipopolysaccharide (LPS), could increase production of IL-23 as well.

24

The result is immune dysregulation that involves increased numbers of Th17 cells and the production of

the pro-arthritogenic and autoimmunogenic cytokine IL-17 [112].

Figure is adapted from DE de Almeida et al: New insights into the functional role of the rheumatoid

arthritis shared epitope. FEBS Letters Vol. 585, Issue 23, 2011, pages 3619–3626.

I.8. T cell epitope mapping study of aggrecan

Cartilage proteoglycan aggrecan (PG) is a large (>2 × 106 daltons) and complex

macromolecule. The core protein of PG consists of ∼2,400 amino acids to which

negatively charged glycosaminoglycan side chains are covalently attached. In an

epitope mapping study using a total of 143 synthetic peptides containing predicted T

cell epitopes, we identified 27 peptide sequences that induced T cell responses in PG-

immunized BALB/c mice. Four of these T cell epitopes were dominant (designated

“arthritogenic”), whereas the others were either subdominant or cryptic [119]. Synthetic

peptides (15 amino acids long) containing the predicted T cell epitopes, alone as a

single peptide or in combination with other peptides (4×1–100 μg of peptide per mouse)

were used for hyperimmunization of BALB/c mice, coimmunization with cartilage PG,

or for eliciting arthritis (inflammation) in mice that had been preimmunized with

cartilage PG (“prearthritic” state) [120, 121]. While positive control groups immunized

with cartilage PG consistently developed arthritis (95–100% incidence), none of the

peptides used in any combinations for immunization induced disease. Some peptides

even induced immunologic tolerance [52].

Since (hyper)immunization with synthetic peptides, including those designated as

arthritogenic, did not induce arthritis in repeated experiments, we administered a single

dose of cartilage PG (100 μg of protein) to peptide-immunized mice 4 weeks after the

fourth (last) peptide injection. This single injection of cartilage PG does not induce

arthritis, and only moderate antibody and T cell responses can be detected 2–3 weeks

after the injection. There was no arthritis in peptide-immunized and PG-challenged

25

BALB/c mice, except in a group that had been hyperimmunized with peptide p135H. Of

10 p135H-hyperimmunized mice, 6 developed arthritis within 9–12 days and 4

developed arthritis within 3 weeks after a single injection of human PG [52].

The p135H peptide sequence (2373

TTYKRRLQKRSSRHP) is located in the C-terminal,

carbohydrate side chain–free, G3 domain of PG, and this peptide was originally

identified as cryptic rather than arthritogenic [11]. Interestingly, p135H shows high

sequence homology with proteins of common human pathogens (e.g. several bacterial

heat-shock proteins) and the “shared epitope”, the most common sequence motif

(QKRAA) in HLA–DR4 alleles, which predispose humans to the development of RA

[52].

26

II. AIMS OF THE STUDY

The common purpose of studying experimentally induced arthritis is to find an animal

model that simulates inflammation, cartilage degradation and immunological events that

are characteristic of human rheumatoid diseases. Proteoglycan-induced arthritis (PGIA)

shares several features with rheumatoid arthritis (RA) and ankylosing spondylitis. The

aim of our studies was to gain more insight into the immunological events behind the

pathologic changes in PGIA.

II.1. Investigation of the role of peptide p135-specific lymphocytes and T cell

hybridomas in the induction of arthritis

The aim of the study was to investigate the immunologic function and determine the

fine epitope structure of a synthetic peptide p135H (2373

TTYKRRLQKRSSRHP) of the

G3 domain of human cartilage proteoglycan (aggrecan), which contains a highly

homologous sequence motif of the “shared epitope” (QKRAA), the most common

sequence motif in HLA-DR4 alleles, which predispose humans to the development of

RA. To further investigate whether the T cell epitope represented by p135H might be

directly involved in the pathogenesis of the disease, and reveal the precise

immunological mechanisms in the background, we performed the following

experimental studies:

1. (Hyper)immunization experiments using p135 and peptide priming of

lymphocytes.

2. Generation of p135-reactive T cell hybridomas.

3. Determination of the peptide specificity and antigen specific cytokine profile of

27

lymph node cells and T cell hybridomas.

4. Characterization of cell surface markers of p135-specific lymphocytes and p135-

reactive T cell hybridomas.

5. Determination of the MHC restriction of T cell hybridomas.

6. Identification of the core epitope and amino acid residues critical for antigenicity

of peptide p135.

7. Determination of the MHC-binding residues of p135H.

8. Identification of T cell receptors of p135-reactive T cell hybridomas.

9. Transfer of arthritis into SCID mice using p135H-primed lymphocytes and

p135H-specific T cell hybridomas.

II.2. Investigation of the adjuvant effect of dimethyldioctadecylammonium bromide

(DDA) in proteoglycan-induced arthritis and collagen-induced arthritis

In an attempt to exclude the effect of bacterial HSPs in autoimmune models of arthritis,

and the Freund’s complete adjuvant (CFA)-induced irritation and granuloma formation

with subsequent adhesions in the peritoneal cavity [93], we used a positively charged

lipophilic quaternary amine, dimethyldioctadecylammonium bromide (DDA) as

adjuvant in PGIA and CIA instead of the conventionally used CFA. PGIA and CIA

were induced using standard immunization protocols with cartilage PGs or human type

II collagen (CII) emulsified with CFA, and compared with PGIA and CIA generated

using immunization protocols in which the same antigens were administered in

combination with the adjuvant DDA. Immune responses to immunizing and self PGs

and CII, the incidence, severity and onset of arthritis were monitored throughout the

experiments.

28

In this study we performed the following experiments:

1. Isolation of antigenic components from cartilage of different species.

2. Immunization of different inbred mouse strains according to the standard

PG/CFA protocol or using DDA as adjuvant.

3. Assessment of arthritis onset, incidence and severity.

4. Measurement of antigen-specific T cell responses, antibodies and cytokines.

5. Characterization of cell surface markers.

6. Cell isolation and transfer of arthritis into SCID mice.

29

III. MATERIALS AND METHODS

III.1. Isolation of antigenic components from cartilage

III.1.1. Crude (total) cartilage extracts

Total cartilage extracts were obtained by 4M guanidinium chloride extraction [49] from

newborn and adult human and bovine articular cartilage, bovine nasal cartilage, ewe and

swine articular cartilage, chicken sternal cartilage, cartilaginous skeletal tissue from

newborn mice and rat chondrosarcoma. These cartilage extracts were dialyzed against

water and lyophilized (hereafter called crude cartilage extract), or further purified by

gradient centrifugation to obtain highly purified cartilage proteoglycans (PG).

III.1.2. High-density cartilage PG

High-density cartilage PG was purified by repeated cesium chloride gradient

ultracentrifugation under dissociative conditions (in 4M guanidinium chloride), as

described in detail elsewhere [49]. Purified cartilage PGs were deglycosylated for

immunization or were left untreated. None of the purified PGs (“native PG”) or the

crude cartilage extracts without deglycosylation induced arthritis, and the results with

deglycosylated cartilage samples are therefore presented (Table 1).

III.1.3. Type II collagen

Type II collagen was purified by sequential and repeated precipitation with NaCl in

0.5M acetic acid at 4°C.

30

Table 1. Arthritogenic effect of different species of cartilage PGs on BALB/c mice and their

immune responses to mouse cartilage PG*

Serum autoantibody to

mouse PG, mean ± SD

mg/ml

Cartilage PG source

(deglycosylation)

Day of

earliest

onset †

Arthritis

incidence ‡

Day of

maximum

incidence §

Cumulative

severity score,

mean ± SD

Proliferation,

SI, mean ±

SD

IgG1 IgG2a

Human newborn articular cartilage

PG (ABC) 26 15/15 (100) 38 11.8 ± 3.8 3.8 ± 1.3 0.5 ± 0.1 0.1 ± 0.0

Human adult articular cartilage PG

(ABC/endo-β-gal) 26 15/15 (100) 46 12.4 ± 1.5 2.7 ± 0.3 0.4 ± 0.2 0.1 ± 0.0

OA-24; human OA cartilage extract

(ABC/endo-β-gal) 26 44/44 (100) 48 9.9 ± 1.7 2.4 ± 1.0 0.3 ± 0.1 0.1 ± 0.1

Human adult articular cartilage PG

(ABC) 42 9/9 (100) 58 8.8 ± 3.4 2.9 ± 2.1 0.3 ± 0.2 0.1 ± 0.0

Canine articular cartilage PG

(ABC) 27 15/15 (100) 47 12.8 ± 1.6 4.7 ± 3.0 0.5 ± 0.2 0.1 ± 0.0

Bovine nasal/articular cartilage PG

(ABC) 47 20/20 (100) 66 8.8 ± 3.2 2.7 ± 0.1 0.1 ± 0.0 ND

Fetal bovine articular cartilage PG

(ABC) 48 9/9 (100) 68 13.5 ± 2.4 2.4 ± 0.2 0.1 ± 0.0 ND

Porcine articular cartilage PG

(ABC/endo-β-gal) 32 9/9 (100) 51 12.2 ± 1.8 2.8 ± 0.5 0.1 ± 0.0 0.1 ± 0.0

Porcine articular cartilage PG

(ABC) 32 4/5 (80) 68 ** 12.8 ± 2.9 2.0 ± 0.2 0.2 ± 0.0 ND

Sheep articular cartilage PG

(ABC/endo-β-gal) 45 5/5 (100) 70 7.8 ± 3.0 2.1 ± 0.3 0.1 ± 0.0 ND

Chicken sternal cartilage PG

(ABC) NA 0/14 (0) NA NA 5.6 ± 2.2 0.1 ± 0.0 ND

Newborn mouse PG NA 0/8 (0) NA NA 1.1 ± 0.1 0.2 ± 0.1 ND

Rat Swarm chondrosarcoma PG

(ABC) NA 0/12 (0) NA NA 1.0 ± 0.2 0.7 ± 0.1 0.1 ± 0.0

PG-free human adult articular

cartilage extract NA 0/10 (0) NA NA 6.4 ± 1.4 ND ND

PBS + DDA NA 0/22 (0) NA NA 0.9 ± 0.2 ND ND

Type II collagen from human adult

articular cartilage NA 0/15 (0) NA NA 3.6 ± 1.4 ND ND

* Mice were immunized with purified cartilage PG aggrecan (100 μg of PG core protein in 100 μl of

phosphate buffered saline [PBS]) and dimethyldioctadecylammonium bromide (DDA; 1 mg/100 μl). The

OA-24 group received crude cartilage extract from osteoarthritic human cartilage (∼100 μg of PG core

protein). Native (nonglycosylated) cartilage PGs did not induce arthritis when used in emulsion with

either of the Freund's adjuvants [3] or with DDA (results not shown). The type of deglycosylation

required to retrieve the arthritogenic potential of a given cartilage PG sample is shown. Chondroitin

sulfate side chains were removed by chondroitinase ABC (ABC) treatment; both chondroitin sulfate and

keratan sulfate side chains were depleted by chondroitinase ABC and endo-β-galactosidase (ABC/endo-β-

gal) treatment. Each animal received 4 injections, regardless of whether arthritis developed at any

immunization time point. Mice were sacrificed at the same time on days 91–92. Experiments were

repeated 2 or 3 times (except those groups with 5 mice), and the results are summarized. Results for the

bovine nasal and bovine articular cartilage are combined. NA = not applicable; ND = not detected.

† The day when the earliest onset of arthritis (first arthritic animal) was diagnosed in the group.

‡ Number of arthritic animals/all immunized animals (%).

§ The day when all mice in the immunized group developed arthritis, except in negative (NA) groups or

in the group labeled with the double asterisk.

Antigen-specific T cell proliferation in the presence of the same antigen as used for immunization (50 μg

protein/ml) expressed as the stimulation index (SI).

31

III.2. Synthetic peptides

Fifteen amino acid long peptides representing the human aggrecan core protein

sequence (p135H) or corresponding sequences of aggrecan from other species, such as

mouse, bovine, canine, and porcine (Table 2), were synthesized by Research Genetics

(Carlsbad, CA). Peptides of p135H having 1-mer offset shifts in sequence or single

amino acid substitutions (Ala or Gly) were synthesized by Chiron Mimotopes (Clayton,

Victoria, Australia). Peptides listed in Table 2 were synthesized in bulk quantities and

purified by high-pressure liquid chromatography (guaranteed purity 90–95%), whereas

pin-peptides with altered amino acid sequences were not purified after cleavage.

Table 2. Amino acid sequences of p135 peptides and their effect on arthritis induction when used

for hyperimmunization of BALB/c mice prior to injection of a single dose of cartilage proteoglycan

Peptide* Origin Sequence† No. of arthritic mice/

no. immunized (%)‡ Arthritis severity score§

p135H Human TTYKRRLQKRSSRHP 14/14 (100) 6.9 ± 3.3

p135M Mouse N***H*****TM*PT 11/15 (73) 3.7 ± 4.8

p135B Bovine A************PL 13/13 (100) 6.8 ± 3.9

p135P Porcine ************SL 9/10 (90) 5.2 ± 2.1

p135D Canine S************A* 12/12 (100) 6.3 ± 2.3

p135H-AA Human-AA **********AA*** 15/15 (100) 7.2 ± 4.3

p130H Human STLVEVVTASTASE 0/9 (0) NA

HLA-DR4 Human KDLLEQKRAAVDTYR 2/9 (22) 1.0 and 2.0

DnaJ Escherichia coli VLTDSQKRAAYDQYG 1/13 (7) 2.0

* All peptides represent amino acid sequences of cartilage PGs, except the Escherichia coli heat-shock

protein–derived DnaJ peptide and the HLA–DR4/Dw4 sequence.

† Asterisks represent amino acids that are the same as those in the corresponding human p135H peptide

sequence. The p135 peptides contain a 5–amino acid motif (underlined) that resembles the “shared

epitope” or “RA cassette” which is present in the conservative amino acid (Ala-Ala)–substituted p135H-

AA sequence. Peptides were used for immunization of BALB/c mice (4 injections of 100 μg of peptide in

CFA followed by the same doses in IFA), which was followed by a single injection (100 μg) of human

cartilage PG in IFA.

‡ The number of arthritic animals in each group and the incidence of arthritis were evaluated 3 weeks

after PG injection.

§ Values are the mean ± SD. NA = not applicable.

32

III.3. Animals

Mice of different strains (see Figure 13A; section IV.2.1., page 60), ages and both sexes

were purchased from the National Cancer Institute (NCI; Frederick, MD), Charles River

Laboratories (Kingston, NY, Raleigh, NY, and Portage, MI colonies), Harlan (Madison,

WI), Taconic Farm (Germantown, NY) and The Jackson Laboratory (Bar Harbor, ME).

All mice were housed in the same room of the Comparative Research Center at Rush

University (Chicago, IL).

Female SCID mice of BALB/c background (NCI/NCrC.B-17-scid/scid) were obtained

from the NCI and maintained under germ-free conditions; these mice were used for

transfer experiments.

III.4. Immunization protocols

III.4.1. Peptide p135 (hyper)immunization experiments and peptide priming of

lymphocytes

Female BALB/c mice were injected intraperitoneally (IP) with 3 different doses of

peptide p135 (1.0 μg, 10 μg, and 100 μg/injection) in CFA followed by similar

injections containing the peptide in IFA on days 21, 42, and 63. Since no joint

inflammation was observed in these p135 peptide–immunized mice after the fourth

injection, peptide-hyperimmunized animals also received a single dose of proteoglycan

(100 μg protein in IFA) IP on day 84.

Female BALB/c mice were injected once into the footpad with 100 μg of human p135

(p135H) or mouse p135 (p135M) peptide in CFA. Mice were sacrificed 9 days later,

and popliteal lymph nodes were harvested. The peptide-primed lymph node cells were

cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% normal

33

syngeneic mouse serum and 5% fetal calf serum (FCS) in the presence of peptide

p135H or p135M (50 μg/ml). Viable lymphocytes were harvested 3 days later by

gradient centrifugation over Lympholyte M (Cedarlane, Hornby, Ontario, Canada) and

further cultured for an additional day in the presence of IL-2. Conditioned medium from

EL-4.IL-2 cells stimulated with 10 ng/ml of phorbol 12-myristate 13-acetate was used

as a source of IL-2. These peptide p135H–specific and peptide p135M–specific in

vitro–restimulated lymphocytes were used for cell transfer into SCID mice or for fusion

to generate T cell hybridomas.

III.4.2. Adjuvant studies: comparison of DDA with CFA

Female and male mice (12–16 weeks of age) of different inbred strains (see Figure 13A;

section IV.2.1., page 60) as well as their F1 hybrids were used to compare selected

immunization protocols. In experiments in which the effects of different cartilage PGs

and/or crude cartilage extracts, with or without treatment of different glycosidases, were

compared (Table 1; page 30 and Figure 12; section IV.2.1., page 60), we immunized

female retired breeder (mature mice that are beyond the age of peak reproductive

performance) BALB/c mice that had been purchased from the NCI. When a new

experimental group was immunized, 10–15 female retired breeder BALB/c mice were

also immunized with deglycosylated human cartilage PGs in DDA or CFA; these

groups were used as positive/reference control groups.

Mice were injected intraperitoneally (IP) with 100 μg of deglycosylated cartilage PGs or

crude extracts (both measured and normalized to PG core protein) or with human

cartilage CII, except where indicated otherwise. A fine cationic liposome form of DDA

(micelle) was obtained by heating a 10-mg/ml DDA suspension (Sigma-Aldrich, St.

Louis, MO) in PBS (pH 7.4) to 56–63°C for 15–20 minutes and then cooling on ice

34

[99]. This DDA micelle form was mixed with an equal volume of antigen (1 mg of

CII/ml or 1 mg of PG core protein/ml of PBS), and the mixture was either shaken for

20–30 minutes at room temperature or vortexed for 15–20 seconds. DDA and

antigen/DDA (CII/DDA or PG/DDA) emulsions were freshly prepared on the day of

immunization and stored on ice until the time of injection. The antigen/DDA micelle

emulsion (200 μl total) was injected IP or, where indicated, subcutaneously (SC) or

intradermally (ID).

Four major groups of animals were treated according to one of the following “standard”

immunization protocols. In protocol 1 (PG/CFA), an IP injection of 100 μg of PG

protein in 100 μl of PBS (pH 7.4) emulsified with 100 μl of CFA (Difco, Detroit, MI)

was given on day 0. On days 21 and 42, the same dose of PG in IFA (Difco) was

injected IP. In protocol 2, the same antigen dose, injection time points, and IP approach

were used, but the 100 μg of PG in 100 μl of PBS was mixed with 1 mg of DDA

micelle in 100 μl of PBS. In protocol 3, 100 μg of CII was dissolved in 0.1M acetic

acid, and the volume was adjusted to 1 mg of CII/ml of PBS to prepare an emulsion

with an equal volume of CFA. The emulsion containing 100 μg of CII was then injected

either IP or ID into the proximal tail on days 0 and 21. In protocol 4, the same dose of

CII (100 μg) in 100 μl of DDA micelle (1 mg) was also used for either IP or ID

immunization.

In addition to these 4 “standard” immunization protocols, we used 5 other approaches.

In protocols 5 and 6, different doses of PGs (protocol 5) or crude cartilage extracts

(protocol 6) were mixed with different amounts of DDA and injected IP, SC, or ID. A

minimum of 0.5 mg of DDA per injection was required to achieve the maximum effect

(incidence and severity) in BALB/c mice, but the onset of arthritis was faster when 1

mg of DDA micelle was used. Higher doses did not change any immune or clinical

35

parameters of arthritis induction, and the IP immunization was the most effective for

inducing arthritis. Concentrations higher than 20–25 mg/ml of DDA micelle were

insoluble, and 3.5–4.0 mg of DDA in 100 μl per mouse per injection in 100 μl resulted

in 15–20% mortality within 4–5 days after IP injection.

In protocol 7, we used PG/DDA in micelle form (heated and cooled as described

above), or water-insoluble DDA simply suspended in PBS at room temperature and

mixed with PG. Although the PG/DDA suspension produced a slightly higher Th1

response and the arthritis onset was 1–3 days earlier than when PG/DDA micelle was

used (data not shown), the PG/DDA suspension required constant shaking, which made

the immunization more complicated and less reproducible.

In protocol 8, we also used purified cartilage PG (100 μg of core protein) in emulsion

with DDA micelle (1 mg) for immunization as a standard method, and we compared the

results with those obtained when the PG and DDA were injected separately into

different areas (e.g., DDA injected IP and PG injected SC or vice versa). IP

administration of DDA appeared to be critical to achieving a maximum adjuvant effect

in PGIA, and ID administration appeared to be critical in CIA. In contrast, IP injection

of DDA was not an irritant, and no granuloma formation or adhesion occurred.

Therefore, unless indicated otherwise, we routinely used 100 μg of PG core protein in

100 μl of PBS with 1 mg of DDA micelle in 100 μl of PBS for IP immunization of

BALB/c mice purchased from NCI.

Along with these preliminary/supplemental experiments, in protocol 9, we also

immunized BALB/c mice with keyhole limpet hemocyanin, ovalbumin, bovine serum

albumin (BSA) or methylated BSA (mBSA) (100 μg of antigen/injection; all from

Sigma-Aldrich) in DDA. Arthritis was scored, and T and B cell responses and cytokine

levels were measured as described below for PG/DDA-immunized animals.

36

III.5. Assessment of arthritis

The paws of peptide p135 specific lymphocyte or T cell hybridoma transferred mice

were examined daily. The paws of all mice in DDA/CFA comparision related

experiments were examined twice weekly until day 21, and then daily thereafter.

Abnormalities due to arthritic changes of the joints were recorded. The appearance of

the first joint swelling was recorded as the time of onset of arthritis. A standard scoring

system based upon swelling and redness (range 0–4 for each paw; maximum possible

score 16 per animal) was used for the assessment of disease severity as described before

[5, 9]. The limbs and spine of arthritic and nonarthritic mice were dissected, fixed,

decalcified, sectioned, and the tissue sections were stained with hematoxylin and eosin

for histopathologic examination.

III.6. Cell lines and culture media

Cell lines BW5147.G.1.4 (T cell receptor α/β+ [TCRα/β+]), A20 (H-2d), CTLL-2 and

EL-4.IL-2 were obtained from American Type Culture Collection (Manassas, VA). The

culture medium for all cell types was Dulbecco’s modified Eagle’s medium (DMEM)

and either heat-inactivated (56°C for 30 minutes) 10% fetal calf serum (FCS; Sigma-

Aldrich, St. Louis, MO) or 1% pooled normal mouse serum (unless indicated

otherwise). All cultures were performed at 37°C in a humidified atmosphere of 5% CO2

in air.

III.7. Generation of p135-reactive T cell hybridomas

Peptide p135H- or peptide p135M-specific lymphocytes were generated as described

above (see section III.4.1., page 32). These lymphocytes were fused with

BW5147.G.1.4 thymoma cells as described elsewhere [8, 122]. Fused cells were

37

seeded into 96-well plates, and hybridomas were selected in medium containing

hypoxanthine/aminopterin/thymidine (Sigma-Aldrich) and 15% FCS. Hybrids were

screened for reactivity to peptides p135H and p135M using irradiated (140 Gy) (JL

Sheperd & Associates, San Fernando, CA) A20 lymphoma cells as APCs. T cell

hybridomas reactive to the corresponding peptide were frozen, and cells that retained

their antigen (p135) specificity after the second testing were cloned by limiting dilution

as described previously [8].

III.8. Measurement of antigen-specific cell proliferation, cytokine secretion and

antibody production

Spleen and lymph node cells were collected at the end of the experiments. Sera were

collected from immunized mice during the immunization period (once or twice each

week from the retroorbital venous plexus) and at the end of the experiments.

To assess cell proliferation, p135H- and p135M-primed lymph node cells (3 × 105

cells/well) or T cell hybridomas (5 × 104 cells/well) were cultured with or without

peptides p135H and p135M or analog peptides (25–50 μg/ml) in quadruplicate wells of

96-well plates for 36 hours. Irradiated A20 B lymphoma cells (105 cells/well) were used

as APCs for T cell hybridomas in the presence or absence of 25–50 μg/ml of p135H,

p135M or analogous peptides.

In the adjuvant comparison study, antigen-specific T cell responses were measured in

quadruplicate samples of spleen cells or PG/adjuvant-primed lymph node cells (3 × 105

cell/well) cultured in the presence of 25 μg of PG protein/ml or 25–50 μg of CII. In both

study antigen-specific IL-2 production was measured in 2-day-old supernatant by

CTLL-2 bioassay. Briefly, 5 × 103 IL-2-dependent CTLL-2 cells/well were cultured

with 100 μl of supernatants for 24 hours, pulsed with 0.5 μCi of 3H-thymidine (Perkin

38

Elmer, Boston, MA) for 18 hours, and then harvested with an automatic cell harvester

(Tomtec, Orange, CT). The radioactivity incorporated into DNA was measured in a

liquid scintillation counter (Wallac, Gaithersburg, MD). The result was expressed as

stimulation index, which was calculated by dividing the counts per minute (cpm) of

peptide- or PG-stimulated cells by the cpm of unstimulated cells.

Production of antigen-specific IFNγ, IL-4, IL-5, IL-10, IL-12, and TNFα (BD

Biosciences, San Diego, CA, or R&D Systems, Minneapolis, MN) was measured in cell

culture supernatants (3 × 106 cell/ml) on day 4 using capture enzyme-linked

immunosorbent assays (ELISA) according to the manufacturer’s instructions. Cytokines

(IFNγ, IL-1β, IL-6, IL-10, IL-12, and TNFα) in the sera of immunized animals were

measured by ELISA at the end of the experiments.

Antigen-specific antibodies were measured by ELISA. Maxisorp immunoplates (Nunc,

Roskilde, Denmark) were coated with human or mouse cartilage PGs (0.1 μg of

protein/well), and the free binding sites were blocked with 1% fat-free milk in PBS.

Sera were applied at increasing dilutions, and levels of both total anti-PG antibodies and

isotypes of PG-specific antibodies were determined using peroxidase-conjugated goat

anti-mouse IgG (Accurate, Westbury, NY) or rat mAb to mouse IgG1 or IgG2a

(Zymed, South San Francisco, CA) as secondary antibodies. Serum antibody levels

were calculated relative to the corresponding mouse IgG isotype standards (all from

Zymed) or mouse serum immunoglobulin fractions (Sigma-Aldrich). The total serum

IgG fraction was determined by ELISA using mouse κ-chain–specific peroxidase-

labeled rat mAb for detection (BioSource, Camarillo, CA).The color reaction was

quantified with an ELISA reader (Coulter, Hialeah, FL) at 405 nm.

39

III.9. Flow cytometric analysis of cell surface markers

The expression of cell surface markers was assayed by flow cytometry. Briefly, either

106 unseparated spleen cells obtained at different time points of immunization, lymph

node cells from antigen-primed mice (100 μg of antigen with 100 μl of CFA or DDA

micelle were injected into the footpad and cells harvested 9 days later), or cells

harvested from peritoneal lavage fluid were incubated with fluorescein isothiocyanate-

or phycoerythrin-labeled, or biotinylated mAbs to CD3, CD4, CD8, CD25, CD28,

CD44 and CD69 (T cell and T cell activation markers), mAbs to T cell receptor

(TCR)α/β, TCRγ/δ and to various TCR Vβ chains, mAb to CD45/B220 (B cell marker),

mAb to CD68 or F4/80 (monocyte/macrophage marker), mAb to Gr-1 (myeloid cell

lineage marker, e.g. neutrophil leukocytes), and mAb to CD11c (activated dendritic cell

marker). Isotype control antibodies labelled with the corresponding fluorescent dyes

were used as negative controls. Antibodies were purchased from BD Biosciences, R&D

Systems or BioSource. Cells were washed twice in PBS containing 1% and 0.02%

sodium azide. The cells labeled with biotinylated primary antibodies were incubated

with streptavidin-R-phycoerythrin (BD Biosciences) or streptavidin-Alexa Fluor 488

(Molecular Probes, Eugene, OR) for an additional 30 minutes at 4°C. After 2 more

washes, the cells were fixed in 2% buffered formalin, and samples were analyzed on a

FACScan flow cytometer using CellQuest software (both from Becton Dickinson, San

Jose, CA). Mean fluorescence intensity values were used to characterize the expression

of the different cell surface markers.

III.10. MHC restriction of p135-reactiveT cell hybridomas

The MHC restriction of p135H-specific hybridomas was determined using mouse anti-

I-Ad (BD PharMingen) and anti-I-E

d (Cedarlane Laboratories) mAbs. Hybridoma

40

cells were cultured with A20 APCs in the presence of peptide p135H or p135M, and

either anti-I-Ad or anti-I-E

d mAb at concentrations of 5–20 μg/ml. The effect of mAb on

IL-2 production was determined with the CTLL-2 bioassay as described above. Isotype-

matched mAbs were used as controls.

III.11. Determination of the core epitope and amino acid residues critical for

antigenicity of peptide p135

To further characterize the critical peptide sequence required for MHC and TCR

binding, we used peptides with a 1-mer offset covering the sequence p2367–2396 to

define the minimum length of the peptide recognized by a p135H-specific T cell

hybridoma H74/4. Subsequently, single-amino acid (Ala or Gly)-substituted peptides of

p135H were used for the identification of the essential residues involved in the

antigenicity of this epitope. Alanine was used because of its non-bulky, chemically

inert, methyl functional group that nevertheless mimics the secondary structure

preferences that many of the other amino acids possess. Glycine was used because it is

the smallest one of the 20 amino acids, not chiral, and can fit into hydrophilic or

hydrophobic environments, due to its minimal side chain of only one hydrogen atom.

In both cases, H74/4 hybridoma cells were cultured with or without the above-

mentioned panels of peptides (20 μg/ml or 40 μg/ml) in the presence of irradiated A20

APCs, and IL-2 secretion in the supernatants was measured by the CTLL-2 assay as

described above.

III.12. Determination of the MHC-binding residues of p135H

All peptides, including those that did not stimulate T cell hybridoma H74/4 cells, were

also tested for their MHC-binding capability. Briefly, A20 lymphoma cells (2 × 105)

41

were incubated with 2-4 μg of biotinylated p135H or synthetic single-residue-

substituted p135H peptides for 2 hours at 37°C in 100 μl of PBS. After 2 washes with

PBS containing 1% FCS, cells were incubated with 1.5 μg of streptavidin-R-

phycoerythrin at room temperature for 30 minutes. The cells were washed again and

analyzed by FACSCalibur (Becton Dickinson). Results were expressed as geometric

mean values calculated by CellQuest Software (Becton Dickinson). Serial dilutions of

nonbiotinylated peptides of identical sequence were used for competitive inhibition or

nonbiotinylated anti-avidin IgG were used as specificity controls.

III.13. Identification of T cell receptors of p135-reactive T cell hybridomas

Total cellular RNAs from T cell hybridomas were isolated using the TRIzol reagent

method according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The

first-strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA

using an oligo(dT) primer and a SuperScript reverse transcription kit (Invitrogen). The

cDNA were used as templates to amplify the transcripts of the Vα and Vβ regions of the

TCR. To amplify unknown Vβ genes expressed by T cell hybridomas, a degenerate

upstream primer (5′-ATGTACTGGTATCAGCAG-3′) was paired with a Cβ

downstream primer (5′-GCCAAGCACACGAGGGTAGCC-3′). For amplification of Vα

genes, a consensus upstream primer (5′-

TGGTACNDVCAGCATCCYGGVGAAGGCC-3′) was used with a Cα downstream

primer (5′-AGCTTTCATGTCCAGCACAG-3′) [123]. After 35 cycles at 94°C for 45

seconds, 60°C for 45 seconds, and 72°C for 2 minutes, polymerase chain reaction

products were extracted from 1.5% agarose gels using Qiaex II gel extraction kit

(Qiagen, Valencia, CA) and sequenced on an ABI 310 Genetic Analyzer (Perkin

Elmer).

42

III.14. Cell isolation and transfer of arthritis

III.14.1. Transfer of arthritis using p135H-primed lymphocytes and p135H-specific T

cell hybridomas

For adoptive transfer experiments, SCID mice were “presensitized” by injection with a

suboptimum amount of spleen cells (insufficient for arthritis induction) from acutely

arthritic BALB/c mice and PG antigen. Unseparated spleen cells (4 × 106) were injected

IP into syngeneic recipient SCID mice, together with 100 μg of PG on day 0, as

described previously [9]. These presensitized SCID mice that received only 1 injection

of spleen cells plus proteoglycan on day 0 were considered a baseline control group and

were used as a reference for all other experimental groups.

The first experimental group received 5 × 106 p135H-specific in vitro-stimulated

lymphocytes from p135H-primed BALB/c mice along with 80 μg of p135H as a second

injection on day 7. The second group received 5 × 106 in vitro-stimulated lymphocytes

from mice primed with irrelevant peptide p130H (1530

STLVEVVTASTASE) along with

80 μg of synthetic p130H. p130H contains a cryptic T cell epitope in the

glycosaminoglycan-attachment region of the core protein of human aggrecan, and

served as a negative control. The third experimental group (subgroups for each p135H-

specific T cell hybridoma) received 1 × 106 hybridoma cells intravenously and 80 μg of

p135H IP on day 7. The positive control group received a second IP injection of 1 × 107

spleen cells from acutely arthritic BALB/c mice on day 7. In each experiment, at least 5

SCID mice per group were used simultaneously, and once the optimum conditions (dose

of antigen or peptide, period of in vitro stimulation of peptide-primed lymphocytes and

number of transferred cells) were determined, transfer experiments were repeated 3–5

times.

43

III.14.2. Transfer of arthritis using spleen cells from PG/CFA or PG/ DDA immunized

mice

Single-cell suspensions were prepared from the spleens of arthritic BALB/c mice.

Donor arthritic mice were immunized with cartilage PG in CFA or in DDA as described

above for protocols 1 and 2 (section III.4.2., page 34). Mononuclear cells were isolated

on Lympholyte M (Zymed) and used for adoptive transfer of arthritis as described

previously [9]. In all transfer experiments, 1.5 × 107 spleen cells were injected IP on day

0 with 100 μg of cartilage PG (measured as protein), and 1 × 107 spleen cells from

arthritic donors were injected on day 7 without cartilage PG into SCID mice. There

were absolutely no differences in the incidence, onset, severity or histopathologic

features of adoptively transferred arthritis in SCID mice that received donor cells from

PG/CFA-immunized [9] or PG/DDA-immunized mice (data not shown).

III.15. Statistical analysis

Statistical analysis was performed using SPSS statistical software (version 10.0.5

(p135) and 7.5 (DDA); SPSS, Chicago, IL). Since the results of all in vitro tests showed

a normal distribution, Student’s 2-sample t-test was used to compare the results from 2

groups in peptide p135 related experiments. Significance was set at P≤ 0.05.

In DDA/CFA immunization experiments the Mann-Whitney and Wilcoxon tests were

used for intergroup comparisons. Both the 5% significance level and the 1%

significance level were used.

44

IV. RESULTS

IV.1. Investigation of the role of peptide p135-specific lymphocytes and T

cell hybridomas in the induction of arthritis

IV.1.1. Induction of arthritis in p135H-immunized BALB/c mice by a single dose of

cartilage proteoglycan

As briefly summarized in the introduction, large-scale experiments were performed to

identify and select arthritogenic epitopes of cartilage PG in genetically susceptible

BALB/c mice. While we were able to identify 4 dominant (arthritogenic) epitopes of a

total of 27 T cell epitopes of the core protein of human cartilage PG, all peptide-

immunization experiments but 1 were unsuccessful in inducing arthritis. A single dose

of human cartilage PG induced arthritis in p135H-preimmunized BALB/c mice. Peptide

p135H, a 15-amino acid sequence in the G3 domain of human aggrecan, was previously

not listed as arthritogenic in BALB/c mice, but shows high sequence homologies with

aggrecan of other species (Table 2; section III.2., page 31).

All BALB/c mice that had been immunized with p135H or analogous aggrecan peptides

of various species and then given a single injection of human PG developed arthritis.

The incidence and severity varied (Table 2), being the lowest in p135M (mouse)

peptide-injected animals. Since the p135H sequence contains a conserved motif

QKRSS, which is similar to the “RA cassette” or “shared epitope” (QKRAA) present in

HLA–DR of patients with RA, as well as to DnaJ from Escherichia coli, we also used

synthetic peptide analogs of p135 with altered sequences. The conservative amino acid

replacement (SS to AA) did not change the arthritogenicity of peptide p135H, while the

“shared epitope” present in either the DnaJ or HLA–DR4 peptide, could presensitize

45

BALB/c mice for arthritis (Table 2). These results confirmed that the p135H sequence,

or peptides containing the “shared epitope”, can prime BALB/c mice, although none of

the peptide-hyperimmunized mice developed arthritis without additional challenge with

proteoglycan.

IV.1.2. In vitro T cell responses in p135H- and p135M-primed BALB/c mice

Since all of the p135 synthetic peptides were able to sensitize BALB/c mice (Table 2),

the next question was whether T cells primed by human p135H synthetic peptide could

cross-react with those primed by mouse (self) p135M peptide. As shown in Figure 3,

the mouse-specific p135M peptide could prime BALB/c mice, and lymphocytes from

either p135H- or p135M-primed mice cross-reacted with analog peptides of different

species. However, none of the p135 peptide-primed lymphocytes could be stimulated

with cartilage PG, indicating the cryptic character of the T cell epitope incorporated into

the p135 sequence. This was the case not only for p135M and p135H peptide-primed

lymphocytes, but also for all other p135 peptides, DnaJ from E. coli, and p135H-AA-

substituted analog peptides listed in Table 2.

46

Figure 3. Antigen (peptide)–specific interleukin-2 production by A, human p135 (p135H)-primed and B,

mouse p135 (p135M)-primed lymph node cells stimulated in vitro with p135 analogous peptides of

different species (see Table 2 for sequences), and human and mouse cartilage proteoglycans (PGs). Cells

were stimulated in vitro with 25, 50 and 100 μg/ml (50 μg/ml is shown) of p135 peptides, or with 50

μg/ml and 100 μg/ml (100 μg/ml is shown) of PGs. The cpm in nonstimulated cultures (3 × 105

cells/well) ranged between 1,200 and 2,400 (broken lines). Bars show the mean and SD stimulation

indices (fold increases in stimulation in the presence of peptide) from 2 independent experiments (n = 8

mice in each experiment). All peptides induced significant T cell proliferation relative to nonstimulated

cultures, but neither human nor mouse cartilage PG stimulated T cells due to the cryptic character of the

p135H/M epitope. ∗ = P < 0.05; ∗∗ = P < 0.01.

IV.1.3. Cell surface markers and cytokine secretion of p135H- and p135M-primed

and in vitro restimulated lymphocytes

Peptide p135-primed lymph node cells were mostly CD4+ cells (63% of the CD3

+ cells)

and were TCRα/β+ (70%). Sixteen percent of the CD3

+ cells were CD25

+, and 30% of

the T cells were Vβ8+. The ratio of CD3

+ T cells (73%) to CD45/B220

+ B

47

lymphocytes (25%) in peptide-primed lymph nodes was very similar in both p135H-

and p135M-primed animals. Peptide-primed CD4+ cells were predominantly of the Th1

type, producing large amounts of IFN-γ, twice as much in p135H-stimulated as in

p135M-stimulated cultures, and only trace amounts of IL-4 (Figure 4). TNF-α was

essentially undetectable, and production of peptide-specific IL-5 and IL-10 was detected

only in lymph node cultures of p135H-primed animals (Figure 4). Taken together, these

data indicate that both self and non-self p135 peptide sequences can elicit T cell

reactions in BALB/c mice, and peptide priming induces a predominant Th1 type of

response.

Figure 4. Cytokine secretion of A, human p135 (p135H)-primed and B, mouse p135 (p135M)-primed

lymph node cells after in vitro restimulation with the corresponding peptides. Lymph node cells were

cultured in medium alone or with 50 μg/ml of peptide p135H or p135M. Supernatants were harvested on

day 4 and assayed for IFN-γ, TNF-α, IL-4, IL-5 and IL-10 concentrations by ELISA. Bars show the mean

of 6 independent experiments; SDs were <5% and are not shown.

48

IV.1.4. Induction of arthritis in SCID mice by p135H peptide-primed lymphocytes

We used in vivo cell transfer to determine which aggrecan epitope-specific lymphocyte

population is capable of inducing arthritis. In a previous study, we transferred syngeneic

lymphocytes to SCID mice to monitor subpopulation recovery and to reconstitute a

pathologic homeostasis required for arthritis induction [9]. To bring the recipient SCID

mice to a prearthritic state and reduce the time needed for the development of arthritis,

mice were “presensitized” by injecting a suboptimum number of spleen cells (4 × 106)

from acutely arthritic BALB/c donors, along with a single dose (100 μg) of human

proteoglycan. Presensitization of recipient SCID mice was particularly useful when T

cell hybridomas with a metastatic behavior were used for the transfer experiments.

SCID mice that received only a single injection of 4 × 106 unseparated spleen cells from

arthritic BALB/c donors did not develop arthritis within 2 weeks after the transfer, and

even later, fewer than 50% of the recipients developed arthritis with very low severity

scores (Figures 5A and B). These presensitized SCID mice were considered baseline

control animals.

49

Figure 5. Incidence and severity of arthritis in SCID mice injected with A and B, human p135 (p135H)-

primed and in vitro-restimulated lymphocytes, and C and D, p135H peptide-specific T cell hybridoma

H74/4, as described in Materials and methods. Initially, all SCID mice (n = 15 in each group) received 4 x

106 unseparated spleen cells from arthritic BALB/c mice together with 100 μg of proteoglycan

intraperitoneally on day 0 (arrow); this is a suboptimum dose and can only presensitize SCID mice. A

group of SCID mice that received only a single injection of splenocytes with proteoglycan was

considered the baseline control (ctrl) in each experiment. On day 7 (arrowhead), SCID mice received a

challenging dose of splenocytes from arthritic mice (positive control), peptide p135H-primed or irrelevant

peptide p130H-primed and in vitro-stimulated lymphocytes from popliteal lymph nodes (A and B), or

peptide p135H-specific T cell hybridoma H74/4 (C and D). The arthritis score represents arthritic animals

only.

Experimental groups (except the baseline control group) were reinjected with 5 × 106

peptide-primed lymph node cells restimulated in vitro for 3 days with the corresponding

synthetic peptide. As summarized in Figures 5A and B, peptide p135H-primed

lymphocytes induced arthritis in all presensitized SCID mice with a relatively high

severity score (mean ± SD 5.0 ± 2.2), which was similar to those receiving 1 × 107 cells

from acutely arthritic BALB/c mice (positive control). In contrast, only 50% of

recipient SCID mice developed arthritis in a mild (score of 1.8 ± 0.8), occasionally

transient form both in the baseline control group and in the group injected with

irrelevant peptide (p130)-primed and in vitro-stimulated lymphocytes (62% incidence

Days after transfer

Days after transfer Days after transfer

Days after transfer

50

with an arthritis score of 2.4 ± 0.5). In summary, these transfer experiments clearly

indicated that peptide p135H is able to activate lymphocytes, making these cells capable

of inducing arthritis in an adoptive-transfer system. It is unclear why the peptide-

immunized BALB/c mice did not develop arthritis, but the cryptic character of the

p135H epitope (Figure 3) might be a likely reason.

IV.1.5. Histopathologic features of arthritis adoptively transferred by p135H-

specific lymphocytes

The histopathologic features of the arthritic joints of SCID mice (Figure 6) were very

similar to and technically indistinguishable from those described for either the primary

[48, 49] or adoptive-transferred [9] forms of PGIA. Accumulation of mononuclear cells

in the synovium was observed at the time of onset of the first clinical symptoms. A few

days later, with the progression of inflammation (redness and swelling), lymphocytes

became more abundant in the synovium and periarticular connective tissues, and

proliferating synovial cells formed a pannus-like structure. The joint space became

enlarged due to the accumulation of the synovial exudates rich in lymphocytes and

neutrophils (Figures 6B and C). At a more advanced stage of arthritis (∼2–3 weeks after

onset), there was damage to the articular cartilage, accompanied by subchondral bone

erosion (Figure 6D). As shown in Figures 5A and B, until day 19, no sign of

inflammation was detected in mice that received only a single injection of cells

(baseline control) or mice that were injected with irrelevant peptide (p130H)-specific

lymphocytes (Figures 5A and 6A).

51

Figure 6. Histopathologic features of arthritis adoptively transferred to SCID mice by human p135

(p135H)-specific lymphocytes. Shown are hematoxylin and eosin-stained sections from the hind paws of

SCID mice that received A, p130H (irrelevant) or B–D, p135H-reactive lymphocytes. A and B, Sagittal

sections of ankle joints. Insets, Hind paws from which the sections were taken. C, Metatarsophalangeal

(MTP) joint dissected on day 4 after arthritis onset. D, MTP joint dissected 2 weeks after arthritis onset.

Lymphocytes are abundant in the synovium (B and C) and periarticular connective tissue, and

proliferating synovial lining cells are seen on the surface of the synovium (B–D). The articular cartilage is

severely damaged (arrowheads), and massive bone erosion can be seen (arrows).

IV.1.6. Characteristics of peptide p135-specific T cell hybridomas

The next important step was to identify the critical T cell epitope within the p135

sequence and determine the TCR and MHC binding sites. To achieve this goal, T cell

hybridomas with uniform and stable expression of p135-specific TCRs were generated

by fusing p135H- or p135M-primed and in vitro-stimulated lymphocytes with BW5147

thymoma cells. Stable p135H- and p135M-specific hybridomas were selected after

several rounds of cloning. The p135H peptide-specific hybridoma H74/4 responded

strongly to stimulation with p135H, and recognized bovine, canine and porcine

52

sequences with only slight variations in the magnitude of response (Figure 7A).

Moreover, hybridoma H74/4 recognized a modified peptide (p135H-AA), where alanine

was substituted for the 2 serine residues to create a sequence incorporating the “shared

epitope” (Figure 7A and Table 2). In contrast to the p135H-specific hybridoma H74/4,

the p135M-specific hybridoma M24/4 did not cross-react with p135 peptides containing

homologous sequences from other species (Figure 7B).

Figure 7. Antigen (peptide)-specific IL-2 secretion by A, human p135 (p135H)-reactive “arthritogenic” T

cell hybridoma H74/4 and B, mouse p135 (p135M)-derived hybridoma M24/4 stimulated in vitro with

species-specific p135 peptides (see Table 2 for sequences), and human and mouse cartilage proteoglycans

(PGs). Cells were stimulated in vitro with 25, 50 and 100 μg/ml (50 μg/ml is shown) of p135 peptides, or

with 50 μg/ml and 100 μg/ml (100 μg/ml is shown) of PGs using irradiated A20 cells as antigen-

presenting cells. The cpm in nonstimulated CTLL-2 cultures (5 × 104 cells/well) ranged between 4,200

and 6,000 (broken lines). Bars show the mean and SD stimulation indices, which represent the

proliferation of IL-2-sensitive CTLL-2 cells in the presence of conditioned medium harvested from

peptide-stimulated T cell hybridomas. Significant cross-reactions were found between the human, bovine,

canine and porcine p135 peptides, but not with the mouse p135 sequence or with cartilage PGs.

Hybridoma H74/4 recognized p135 peptides of various species, but did not respond to peptide p135M or

cartilage PGs. Reciprocally, hybridoma M24/4 was stimulated only by peptide p135M. ∗ = P < 0.05; ∗∗ =

P < 0.01.

53

While both p135H- and p135M-specific hybridomas produced relatively high levels of

IFN-γ and TNF-α in response to peptide-specific stimulation, the p135M-specific

hybridomas also produced significant amounts of IL-5 and IL-10 (Figure 8). This

difference might be the reason why the p135M-specific hybridomas did not transfer

(induce) arthritis in SCID mice (see below).

Figure 8. Cytokine secretion by A, “arthritogenic” human p135 (p135H)–specific hybridoma H74/4 and

B, mouse p135 (p135M)–specific hybridoma M24/4 after in vitro stimulation with the specific peptide.

Hybridoma cells were cultured in medium alone, or with 50 μg/ml of p135H or p135M in the presence of

irradiated A20 cells as APCs. Supernatants were harvested on day 4 and then assayed for IFN-γ, TNF-α,

IL-4, IL-5 and IL-10 by ELISA. Bars show the mean of 6 independent experiments; SDs were <5% and

are not shown.

All stable T cell hybridomas, including H74/4 and M24/4, were tested in vivo for

arthritis induction, as described for peptide-stimulated lymphocytes. The only difference

was that T cell hybridomas were injected intravenously, rather than IP, because of their

high metastatic propensity involving the liver. Peptide p135H-reactive T cell

hybridomas (H74/4 was used as a prototype for more detailed characterization) induced

54

arthritis consistently in “presensitized” SCID mice, although both the incidence and

severity of the disease were lower (70% incidence; severity score of 4.29 ± 3.2) in

hybridoma-injected SCID mice (Figures 5C and D) than in those injected with peptide

p135H-primed and in vitro-stimulated lymphocytes (Figures 5A and B), or in those that

received PG-stimulated lymphocytes from arthritic donor BALB/c mice [9].

T cell hybridoma H74/4 was CD4+ and demonstrated high levels of the TCRα/β/CD3

complex, with predominant expression of Vβ10 and Vα1.2 transcripts. This T cell

hybridoma secreted significant amounts of IL-2, TNF-α and IFN-γ, but not IL-4 upon

peptide p135H stimulation (Figure 8A), indicating that it belonged to the Th1 subset.

Recognition of peptide p135H by hybridoma H74/4 was class II MHC-restricted.

Peptide p135H was presented in the context of I-Ed molecules, since a mAb against I-Ed

completely abrogated the p135H-specific in vitro IL-2 secretion of the H74/4

hybridoma (data not shown).

IV.1.7. Determination of the core sequence of peptide p135H that is critical for

recognition by T cell hybridoma H74/4

To determine the fine epitope sequence of arthritogenic T cell hybridoma H74/4

(representing a p135H-specific immortalized lymphocyte), we first examined the

minimal core peptide required to stimulate H74/4 hybridoma cells using a set of

overlapping peptides with a 1-mer offset (p2367–2396 of human aggrecan). A murine B

cell lymphoma cell line (A20) was used as APCs, and IL-2 production was measured

with the CTLL-2 bioassay. The responses of T cell hybridoma H74/4 to the synthetic

peptides are shown in Figure 9. These results define amino acids 2374–2382

(TYKRRLQKR) as the core region containing the minimal sequence required for the

activation of T cell hybridoma H74/4.

55

Figure 9. Mapping of amino acid residues in the core epitope critical for recognition by human p135

(p135H) peptide-specific T cell hybridoma H74/4. A set of overlapping synthetic peptides with a 1-mer

offset was used (peptides p2367–2390 are shown). Irradiated A20 cells were used as APCs, and IL-2

secretion was determined by CTLL-2 bioassay. Bars show the mean stimulation index in triplicate wells

from 3 independent experiments (n = 9) at 2 peptide concentrations; SDs were <5% and are not shown.

Underlined sequences represent the core peptide sequence; asterisk indicates the wild-type peptide

(p135H) used for priming.

IV.1.8. Stimulatory activity of single-amino acid-substituted altered peptides

To study the functional role of each residue in the core sequence of peptide p135H, the

stimulatory activities of analog-altered peptides were tested on peptide p135H–specific

T cell hybridoma H74/4. Alanine and glycine substitutions of residues at positions P1,

P7 and P9 had no major effect on the stimulatory activity compared with the wild-type

peptide p135H (Figure 10). However, substitution of a single amino acid at the P2–P6

or P8 positions completely abrogated the T cell response, indicating that these residues

are likely to be critical for recognition by T cell hybridoma H74/4.

56

Figure 10. IL-2 production by T cell hybridoma H74/4 in response to the wild-type peptide (human p135

[p135H]) and peptides altered by single-amino acid substitution (Ala or Gly). IL-2 secretion was

measured by CTLL-2 bioassay. Bars show the mean stimulation index in triplicate wells from 3

independent experiments at 2 peptide concentrations; SDs were <5% and are not shown. The single-letter

amino acid codes indicate the site of each substitution within the core peptide. The pair of bars at the top

represents the non-substituted wild-type peptide.

IV.1.9. Localization of the I-Ed-binding residues within p135H

To define which residues within p135H were involved in the interaction with I-Ed

molecules on the A20 antigen-presenting cells, we analyzed the I-Ed binding capacity of

single-amino acid (Ala or Gly)-substituted peptide analogs (described above) using flow

cytometry. The results of this analysis (i.e., testing the binding of peptides representing

the core region) are shown in Figure 11. Although most of the substitutions were well

tolerated at the level of MHC binding, 4 substitutions led to a marked decrease in

peptide binding to the I-Ed molecule. The 4 affected substitutions were mostly basic

residues at positions P2 (Tyr), P3 (Lys), P4 (Arg) and P5 (Arg). This corresponds to the

results of other studies that also found that centrally located basic amino acid plays a

major role in the binding of peptides to I-Ed [124, 125]. The positively charged residues

in p135H could possibly interact with the negatively charged residues at positions 114

(Glu) and 155 (Asp) of the Ed

β-chain. With the exception of the P1, P7 and P9

substitutions that had no appreciable effect on T cell stimulation (Figure 10),

57

substitutions at all other residues (P2 [Tyr], P3 [Lys], P6 [Leu] and P8 [Lys]) appeared

to interfere with recognition by the TCR.

Figure 11. Determination of MHC-binding residues within peptide p135H using A20 cells (H-2d) and 4

μg of biotinylated single-amino acid (Ala or Gly)-substituted synthetic peptide variants. A double-layer

avidin-biotin/fluorescein isothiocyanate method was used, and the binding of peptides to H-2d was

assayed using flow cytometry. Results are expressed as percentages of the geometric mean values

normalized to non-substituted, wild-type (WT) p135H (100%). Bars show the mean of 5 independent

experiments; SDs were <10% and are not shown. Alanine- or glycine-substituted amino acids of the core

peptide are indicated with single-letter codes. ∗ = P < 0.05; ∗∗ = P < 0.01.

58

IV.2. Investigation of the adjuvant effect of

dimethyldioctadecylammonium bromide in proteoglycan-induced

arthritis and collagen-induced arthritis

IV.2.1. Incidence and severity of PGIA in arthritis-susceptible BALB/c and C3H

colonies

DDA was first used in our laboratory when we studied how heat shock proteins (HSP)

affect the onset and severity of PGIA [126], since we wanted to avoid the undesired

effects of the bacterial HSPs present in CFA. A group that received proteoglycan with

DDA (PG/DDA) intraperitoneally, developed more severe PGIA significantly earlier

than did BALB/c mice immunized with the same dose of PG with CFA and IFA (Figure

12).

PGIA can only be induced in genetically susceptible BALB/c or C3H strains, but since

the severity and susceptibility can vary even between different colonies of the same

murine strain, we compared the major clinical parameters (arthritis onset, severity and

incidence) in commercially available BALB/c [49] and C3H colonies [53]. While the

intergroup (substrain) differences (50–100% incidence with arthritis scores of 5.1–12.4)

occurred in BALB/c mice, the individual differences among BALB/c colonies

disappeared by week 9 in PG/DDA-immunized mice (data not shown). The incidence

was 100%, and the mean ± SD arthritis score in 9 different BALB/c colonies (n = 462)

was 10.6 ± 3.6 at week 9 (Figure 13B).

59

Figure 12. Incidence and severity of PGIA in BALB/c mice immunized with human cartilage

proteoglycan aggrecan (PG) in Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA),

or dimethyldioctadecylammonium bromide (DDA). Human PG was deglycosylated of both chondroitin

sulfate and keratan sulfate side chains (see Materials and methods). A, The incidence of arthritis was

assessed twice a week. B, Each paw of each animal was scored for inflammation (0–4 scale) resulting in

an arthritis score for each immunized animal (range of possible scores 0–16). Arrows mark the injection

times for PG/CFA (solid arrows), PG/IFA (shaded arrows), and PG/DDA (open arrows with solid

arrowheads). The cumulative results of 4 independent experiments (n = 10–15 animals per experiment)

are shown.

60

Figure 13. Comparative analysis of 9 BALB/c substrains, 3 C3H substrains, F1 hybrids of BALB/c ×

DBA/2 (CDF1) and an additional 10 inbred strains with different H-2 haplotypes. Female mice were

immunized intraperitoneally with deglycosylated human cartilage PG in DDA as shown in Figure 12,

except that all animals, regardless of their stage of arthritis, received 4 PG/DDA injections. All animals

were sacrificed on days 91–92 of immunization (4 weeks after the fourth antigen injection).

61

Data from the end of the experiments in all animals, whether arthritic or non-arthritic, are shown (n = 6–

16 animals per group). A, Inbred mouse strains that were studied. B, Incidence of arthritis and arthritis

scores. C, Antigen-specific T cell responses: proliferation (expressed as stimulation index [SI]),

production of IFN-γ and IL-4. D, Serum level of heteroantibodies, autoantibodies and the IgG1:IgG2a

ratios in PG/DDA-immunized mice. Significant differences between groups or pooled groups are

indicated: ∗ = P < 0.05; ∗∗ = P < 0.01. The corresponding MHC haplotype of each strain is shown across

the bottom of A and B (panel 1). In each panel, the groups are shown in the same order as in A. Values

are the mean and SD. See Figure 12 for other definitions.

In contrast to the BALB/c strain, the extreme differences among C3H colonies, for

example, between C3H/HeJ and C3H/HeJCr, 2 colonies which are otherwise derived

from the same founder [53], remained significant in PG/DDA-immunized mice

(compare columns 4 and 5 in Figure 13B). Only 28% of the C3H/HeJ mice (from The

Jackson Laboratory) developed arthritis with very low arthritis scores (1.9 ± 1.2), after

up to 4 antigen injections (column 5, Figure 13B, panel 2), whereas the incidence was

100% in C3H/HeJCr mice (from the NCI) with an arthritis score of 5.7 ± 2.3 (column 4,

Figure 13B).

Although all BALB/c mice and the highly susceptible C3H colonies uniformly reached

100% incidence of arthritis by weeks 9-10 in response to PG/DDA immunization

(Figure 13B, panel 1), there were significant differences in the final arthritis scores in

BALB/c mice (arthritis score 11.6 ± 1.6) compared with the highly susceptible C3H

colonies (arthritis score 7.2 ± 2.1) (Figure 13B).

IV.2.2. PGIA susceptibility in inbred murine strains

Susceptibility to PGIA is determined by both MHC and non-MHC genes [3, 127], and

the different colonies of susceptible BALB/c (H-2d) and C3H (H-2

k) strains exhibit

variabilities in both the severity and incidence of PGIA (Figure 13B). Therefore, the

next evident question was whether other inbred murine strains with the same (H-2d or

H-2k) or different haplotypes can develop PGIA in response to PG/DDA immunization.

While all murine strains, regardless of their H-2 haplotype or genetic background,

62

responded well to PG/DDA immunization (Figures 13C and D), we could not find any

strain other than BALB/c and C3H that was susceptible to PGIA (Figure 13B, panel 1).

This was especially interesting because a number of murine strains carried the same

class II alleles (either H-2d or H-2

k haplotype), and exhibited similar, or occasionally

even higher T cell and B cell responses to PG immunization, compared with the

susceptible BALB/c or C3H colonies (Figures 13C and D).

IV.2.3. Arthritogenic effect of immunization with DDA and PGs isolated from

various species

In previous studies, we tested cartilage PGs from various species for their ability to

induce arthritis in BALB/c mice when used with Freund's adjuvants [3, 48]. PGs from

fetal human and pig cartilage, and from adult human and canine cartilage were the only

ones that could induce arthritis in BALB/c mice, but only if the glycosaminoglycan

(chondroitin sulfate, or both chondroitin sulfate and keratan sulfate) side chains were

removed [10]. Cartilage PGs from other species either did not induce arthritis or

induced only a weak and transient inflammation at a very low incidence (<10%) in

BALB/c mice immunized with PG in Freund's adjuvants.

Using DDA as adjuvant, we retested a few, relatively easily accessible cartilage PGs of

various species. Quite unexpectedly, PGs from fetal and adult bovine cartilage, and

from adult pig and sheep cartilage proved as arthritogenic as PGs isolated from newborn

or adult human cartilage, when they were injected with DDA. Moreover, crude cartilage

extract from osteoarthritic cartilage proved to be excellent arthritogenic material if both

the chondroitin sulfate and keratan sulfate side chains of PG were removed [49]; it was

as effective as the highly purified human cartilage PG (Table 1; section III.1., page 30).

Thus, when using lipophilic adjuvant DDA for immunization, PGs with relatively

63

poor arthritogenicity in Freund's adjuvants could induce arthritis with maximum

incidence and high severity in mice of the BALB/c strain (Table 1). While cartilage

macromolecules other than PG in crude extracts have also provoked immune responses,

these molecules of PG- and link protein-free cartilage extracts were not arthritogenic in

BALB/c mice (see PG-free extract of human articular cartilage, Table 1).

IV.2.4. Histopathologic features of the peripheral joints and spine of PGIA-

susceptible mice

In the mice immunized with PG in DDA, the clinical scores for the paws corresponded

well to the histopathologic abnormalities in the small peripheral joints, as described for

the “classic” PG/CFA-induced form of PGIA [3, 5, 48]. Joint inflammation started with

mononuclear (mostly lymphocyte) and polymorphonuclear infiltration, which was soon

accompanied by massive cartilage degradation, followed by bone erosion (Figures 14B–

D).

64

Figure 14. Clinical appearance, histopathologic and radiographic features of PGIA in BALB/c mice

immunized with PG/DDA. A, Normal ankle joint. B, Heavily inflamed ankle joint 2–3 weeks after the

onset of arthritis. Insets, Photographs of the hind paws from which the respective tissues were obtained.

C and D, Higher-magnification views of tarsometatarsal joints at 2 weeks and 4 weeks, respectively, after

arthritis onset. In the acute/subacute phase of the disease, the same joint may have massive lymphocyte

and neutrophilic granulocyte infiltration, as well as extensive cartilage and bone erosions (B–D). The

erosions are the consequences of invasion by the aggressive pannus-like synovial tissue (pannus).

Shaded arrows with white arrowheads in C indicate the borders between bone and bone-resorbing soft

tissue (pannus). Arrowheads in C, D and H indicate areas of cartilage erosion. E, Normal knee joint.

Jsp = joint space. F, Knee joint obtained on the day of arthritis onset, showing very early inflammation,

as indicated by thickening of the synovial lining cell layer (parallel arrows). G and H, Knee joints

obtained 4 days and 12 days, respectively, after arthritis onset. Arthritis onset was recorded as paw

inflammation. Dotted lines in G and H, and arrowheads indicate cartilage erosions. Curved white

arrow in G indicates proliferation of a pannus-like granulomatous tissue at the bone–cartilage junction.

I–K, Representative micrographs of intervertebral discs from a normal, non-immunized animal (I), and

from mice with PGIA (J and K). J, Histologic features of a relatively acute (early) onset of spondylitis

(∼2–3 weeks after the onset of inflammation in the peripheral joints) with hyperproliferative pannus-like

granulation tissue (curved white arrows). This aggressive, pannus-like tissue destroys the annulus

fibrosus and resorbs the nucleus pulposus. K, Histologic features of a completely resorbed intervertebral

disk, with segments of vertebrae ankylotized by fibrotic tissue and chondrophytes, ∼8–10 weeks after the

onset of peripheral joint inflammation. Insets, Radiographs of the spines from which the respective

tissues were obtained. All tissue sections were stained with hematoxylin and eosin. See Figure 12 for

other definitions.

65

While these histopathologic abnormalities were very similar to those previously

described in either BALB/c or C3H mice immunized with PG in Freund's adjuvants [5,

49, 53], comparison of the 2 immunization protocols revealed remarkable differences in

the knee joint (Figures 14E–H) and spine (Figures 14I–K). For example, inflammation

usually can be seen in the ipsilateral knee joint 2–3 weeks after the onset of paw

inflammation (small joints) in PG/CFA-immunized BALB/c mice. In contrast, both

small joints and ipsilateral large joints (Figures 14F and G) were simultaneously

inflamed in PG/DDA-immunized mice, but the periarticular inflammation (edema and

cellular infiltration) was less pronounced in the large joints. A second remarkable

difference was that simultaneously with the synovial joint inflammation, massive

spondylitis was detected in all arthritic BALB/c mice that had been immunized with

PG/DDA (Figures 14J and K). This was especially unusual, because spondylitis

typically only appeared 2–4 months after peripheral (synovial) joint inflammation in

BALB/c mice that had been immunized with PG/Freund's adjuvants.

IV.2.5. Mechanisms of action of DDA as adjuvant

As briefly summarized in Materials and methods, a large number of preliminary and

supplemental experiments (protocols 5-9) were performed to determine the optimum

conditions (dose, combination and route) of PG/DDA immunization for induction of

PGIA. The overall results of T cell- and B cell-mediated immune responses to human

PGs in PG/CFA- or PG/DDA-immunized BALB/c mice, however, were highly

comparable, and serum levels of proinflammatory cytokines were even more

pronounced in PG/CFA-immunized animals than in those injected with PG/DDA (Table

3).

66

Table 3. Immune responses and antigen-specific cytokine production by spleen cells after

immunization of BALB/c mice with cartilage PG in CFA or in DDA*

After first injection After second injection After third injection

PG in CFA PG in DDA PG in CFA PG in DDA PG in CFA PG in DDA

T cell proliferation, SI 2.1 ± 0.25 4.25 ± 0.63† 3.42 ± 1.06 3.21 ± 0.95 3.49 ± 1.13 3.46 ± 1.10

Serum antibody level, mg/ml

IgG1 to human PG <0.02 ND 6.62 ± 3.90 3.36 ± 1.42† 10.6 ± 0.42 9.6 ± 1.52†

IgG2a to human PG <0.02 <0.02 0.09 ± 0.04 0.06 ± 0.05 0.24 ± 0.06 0.38 ± 0.12

IgG1 to mouse PG <0.02 0.10 ± 0.01 0.28 ± 0.18 0.06 ± 0.04† 0.65 ± 0.13 0.45 ± 0.09†

IgG2a to mouse PG ND ND 0.03 ± 0.00 0.02 ± 0.01 0.03 ± 0.00 0.05 ± 0.00

Serum cytokine level, pg/ml

IL-1β 28.5 ± 12.3 10.8 ± 0.11‡ 20.7 ± 0.22 15.0 ± 2.60† 23.7 ± 5.6 19.7 ± 0.16†

TNF-α 77.3 ± 28.4 47.2 ± 12.3‡ 405 ± 55.4 239 ± 23.4‡ 587 ± 44.2 441 ± 55.7†

IL-6 98.6 ± 11.3 72.9 ± 18.3† 101 ± 34.6 86.6 ± 19.6‡ 158 ± 28.2 118 ± 18.3‡

IL-10 ND 10.9 ± 0.09 10.1 ± 12.4 3.5 ± 0.11 4.2 ± 1.8 5.4 ± 0.08

Antigen-specific cytokine production

by spleen cells, pg/106 cells/ml

IFN-γ ND 55.0 ± 9.6 12.1 ± 4.3 55.0 ± 9.40 46.8 ± 12.1 107 ± 32.4

IL-4 ND ND 41.2 ± 5.8 48.2 ± 13.2 25.8 ± 9.7 34.8 ± 8.6

IFN-γ:IL-4 ratio NA NA 0.29 1.14‡ 1.81 3.07‡

IL-12 ND ND ND 11.2 ± 2.60 ND 23.0 ± 11.3

TNF-α ND ND 9.28 ± 0.9 1.20 ± 0.02† 10.0 ± 4.72 6.40 ± 0.91†

Ratio of lymphocytes in spleen

CD3+:B220+ NE NE NE NE 0.83 ± 0.12 0.81 ± 0.06

CD4+:CD8+ NE NE NE NE 0.95 ± 0.02 1.13 ± 0.04†

* Assays were performed 9-11 days after the intraperitoneal injection. Values are the mean ± SD. PG =

proteoglycan aggrecan; CFA = Freund's complete adjuvant; DDA = dimethyldioctadecylammonium

bromide; SI = stimulation index; ND = not detected, or the same amount was measured in non-stimulated

and PG-stimulated spleen cell cultures; IL-1β = interleukin-1β; TNF-α = tumor necrosis factor α; IFN-γ =

interferon-γ; NA = not applicable; NE = not evaluated.† P < 0.05 versus PG in CFA-immunized group.

‡ P < 0.01 versus PG in CFA-immunized group.

Although some studies indicate that the use of DDA for vaccination provokes a more

dominant delayed-type hypersensitivity reaction, accompanied by an antibody response

that is higher than that obtained with the use of many other adjuvants, including CFA

[103], we could not confirm those observations. The serum antibody levels to either PG

or CII, or against a number of arthritis-irrelevant antigens (such as keyhole limpet

hemocyanin, BSA or ovalbumin; data not shown) were similar or even lower when

injected with DDA than with CFA. In contrast, we found extensive differences in

antigen-specific IgG1:IgG2a, and IFN-γ:IL-4 ratios when the 2 adjuvants were

compared throughout the immunizations (Tables 3 and 4). Again, while the overall

effects of DDA and CFA on the T cell response (measured as PG-specific T cell

67

proliferation and IL-2 production) were highly comparable (Table 3), antigen (PG)-

specific cytokine production by either PG-primed spleen cells (Table 3) or peripheral

lymph node cells (data not shown) was significantly different, and was clearly shifted

toward Th1 type immune response.

Table 4. Incidence, severity and immune responses in DBA/1J mice immunized with human CII in

CFA or in DDA*

ID injection ID injection IP injection

CII in CFA,

males

CII in DDA,

males

CII in CFA,

females

CII in DDA,

females

CII in CFA,

males

CII in DDA,

females

In vitro T cell proliferation in the

presence of 25 μg/ml of human CII

Stimulation index 3.4 ± 1.0 2.9 ± 0.8 2.7 ± 1.0 2.4 ± 1.6 3.1 ± 1.6 2.3 ± 1.6

Serum anti-mouse CII antibodies, μg/ml

IgG1 407 ± 28 490 ± 133 398 ± 54 452 ± 78 426 ± 114 341 ± 222

IgG2a 48 ± 17 23 ± 14 66 ± 21 53 ± 22 19 ± 11 20 ± 9.1

Clinical symptoms

Day of earliest onset 30 30 30 30 40 32

Maximum arthritis score 5.8 ± 3.4 8.2 ± 2.2† 5.2 ± 2.4 8.3 ± 3.2† 2.4 ± 0.6 2.4 ± 1.2

Maximum incidence, % 85 100† 90 100 25‡ 25‡

*, † DBA/1J mice were immunized with 100 μg of human type II collagen (CII) emulsified with 100 μl of

Freund's complete adjuvant (CFA) or with 100 μl of dimethyldioctadecylammonium bromide (DDA;

micelle form) and injected intradermally (ID) into the proximal tail or intraperitoneally (IP). Mouse CII

was used for measuring autoantibodies; human CII was used for measuring T cell proliferation. Values

are the mean ± SD of 2 independent experiments (n = 20 mice per group). † P < 0.05 versus group

injected with CII in CFA. ‡ P < 0.01 versus each of the groups injected intradermally.

To gain insight into the local mechanisms of DDA action and to understand why DDA

supported PGIA more powerfully, BALB/c mice were injected IP with PBS, PBS/IFA,

PBS/CFA or PBS/DDA, with or without cartilage PG antigen, and cells obtained from a

peritoneal lavage and from the spleen were harvested at 6 hours, 12 hours, every 24

hours thereafter until day 9, on days 14 and 21 after the first injection, and 9 days after

the second or third injection. As expected, the cell number was significantly higher, and

reached a peak after 24-48 hours, in the peritoneal lavage fluid from all adjuvant-

injected groups compared with the PBS- or PG/PBS-injected groups, and these levels

never returned to normal (data not shown). The cell influx contained predominantly

68

neutrophilic granulocytes (up to 70-85%), and the ratio of the neutrophils was

consistently highest in the CFA-injected groups at every time point evaluated. The cell

number in peritoneal lavage fluid from DDA-injected groups was approximately two-

thirds to one-half the cell number in fluid from CFA-injected animals by days 9-14

(data not shown). The cells consisted almost exclusively of F4/80+ macrophages (mean

± SD 79 ± 11%) and CD3+ T cells (7.0 ± 2.6%), and more than 60% of the CD4

+ cells

were activated (CD4+/CD69

+). Moreover, the ratio and the total number of CD11c

+ cells

(dendritic cells) in the peritoneal lavage fluid were highest in the PG/DDA-injected

group (data not shown).

IV.2.6. Application of DDA in the induction of collagen-induced arthritis

Induction of PGIA required intraperitoneal immunization of genetically susceptible

strains of mice with deglycosylated cartilage PGs, and the synergistic adjuvant effect of

DDA was significantly higher than the effect of even the most powerful batch of CFA.

Therefore, the question of whether DDA could be used to replace CFA in other

frequently used arthritis models was evident. We obtained the same results using

methylated BSA (mBSA)/DDA immunization for antigen-induced arthritis (data not

shown), as described previously for mBSA/CFA immunization [128].

The most important data for collagen-induced arthritis (CIA) are summarized in Table

4. The onset of CIA was highly comparable in various groups of DBA/1 mice, but

100% incidence was reached only in CII/DDA-immunized mice after 2 antigen

injections, with a significantly higher arthritis score in CII/DDA-immunized mice than

in CII/CFA-immunized mice (Table 4). The route of immunization (ID versus IP),

however, was a critical component of arthritis induction (Table 4), and the incidence

reached only 60% even after the third IP injection of CII given either in CFA or DDA

69

(data not shown). It is important to note here that although both murine strains (BALB/c

and DBA/1) showed immune response to immunization with either human PG or CII,

DBA/1 mice did not develop arthritis in response to PG/DDA immunization (data not

shown), and BALB/c mice did not show signs of arthritis after CII/DDA (Table 1;

section III.1., page 30) or CII/CFA immunization [49].

70

V. NOVEL FINDINGS

V.1. Investigation of the role of peptide p135-specific lymphocytes and T cell

hybridomas in the induction of arthritis

1. Following a T cell epitope mapping study of human cartilage proteoglycan

(aggrecan), peptide p135H (2373

TTYKRRLQKRSSRHP), located in the G3

domain of human aggrecan and containing a highly homologous sequence motif

of the “shared epitope” (QKRAA), could induce arthritis in BALB/c mice after a

single injection of human proteoglycan. It is important to note, that a single

injection of cartilage proteoglycan does not induce arthritis.

2. T cells primed by human p135H synthetic peptide could cross-react with those

primed by mouse (self) p135M peptide. However, none of the p135-primed

lymphocytes could be stimulated with cartilage proteoglycan, indicating the

cryptic character of the T cell epitope incorporated into the p135 sequence.

3. Peptide p135-primed lymph node cells were mostly CD4+ cells and were

TCRα/β+, producing large amounts of IFN-γ, indicating that both self and non-

self p135 peptide sequences could elicit T cell reactions in BALB/c mice, and

peptide priming induced a predominant Th1 type of response.

4. Using an adoptive-transfer system, p135H-primed lymphocytes induced

arthritis in “presensitized” SCID mice, with a relatively high incidence and

severity score. The histopathologic features of the arthritic joints of SCID mice

were indistinguishable from those described for either the primary or adoptive-

transferred forms of PGIA. It is unclear why the p135H-immunized BALB/c

mice did not develop arthritis before, but the cryptic character of the p135H

epitope might be a likely reason.

71

5. Applying p135H-specific T cell hybridoma H74/4, we determined the fine

epitope structure of p135H. We defined amino acids 2374–2382

(TYKRRLQKR) as the core region of the peptide. Recognition of p135H by

hybridoma H74/4 was class II MHC-restricted, since p135H was presented in

the context of I-Ed molecules. Centrally located basic amino acids at positions P2

(Tyr), P3 (Lys), P4 (Arg) and P5 (Arg).proved to be responsible for binding of

p135 to I-Ed, while residues P2 (Tyr), P3 (Lys), P6 (Leu) and P8 (Lys) appeared

to play a role in the recognition by the TCR. Peptide p135-reactive T cell

hybridoma H74/4 belonged to the Th1 subset, and induced arthritis consistently

in “presensitized” SCID mice, although both the incidence and severity of the

disease were lower than in those injected with p135H-primed and in vitro-

restimulated lymphocytes.

V.2. Comparison of the adjuvant effects of dimethyldioctadecylammonium bromide

(DDA) and Freund’s complete adjuvant (CFA) in proteoglycan-induced arthritis and

collagen-induced arthritis

1. The overall immune responses (antibody production and antigen-specific T

cell responses) were highly comparable when human PGs in CFA or in DDA

were used. The notable differences were as follows:

2. In PG/CFA-injected mice significantly higher levels of serum

proinflammatory cytokine and PG-specific IgG1, but not IgG2a were measured.

3. In PG/DDA-immunized mice significantly higher ratios of antigen-specific

IFN-γ to IL-4 and significantly lower ratios of IgG1 to IgG2a were observed

than in PG/CFA-immunized mice, indicating a more pronounced shift of the

Th1/Th2 balance toward a Th1 type of response in PG/DDA-injected mice.

72

4. A highest ratio of activated CD3+, CD44

High cells in PG/DDA-primed lymph

nodes; an increased ratio of CD4+ to CD8

+ cells in the spleens of arthritic mice

but not in PG/DDA-primed lymph nodes, and a significantly increased number

of CD4+/CD69

+ cells in peritoneal lavage fluid from PG/DDA-primed BALB/c

mice, were detected.

5. Moreover, at least a 2-4-fold increase in macrophage influx into the peritoneal

cavity, accompanied by more CD11c+ dendritic cells but significantly fewer

polymorphonuclear cells, was characteristically observed in DDA-injected mice.

6. The use of DDA accelerated the development of a more severe arthritis, and

suboptimum doses of cartilage PG or CII antigens, or PGs having only a

suboptimal arthritogenic effect when injected with CFA, were able to induce

inflammation, via a more potent activation of the innate immunity.

73

VI. DISCUSSION

Proteoglycan (aggrecan) has been shown to induce progressive polyarthritis in

genetically susceptible BALB/c mice [5, 48, 49]. Although the underlying mechanisms

are still unclear, T cells, particularly CD4+ lymphocytes, seem to play a crucial role in

the initiation of the disease [6-9, 61]. Our earlier studies explored numerous T cell

epitopes along the core protein of human aggrecan, most of them being located within

the poorly glycosylated globular G1 domain [10, 119]. Interestingly, one of the epitopes

present in the G3 domain at the C-terminus of the human proteoglycan also appeared to

be involved in arthritis induction. Hyperimmunization of BALB/c mice with synthetic

peptide (p135H) representing a segment of the human aggrecan G3 sequence

2373TTYKRRLQKRSSRHP, followed by the injection of a single dose of proteoglycan,

induced progressive polyarthritis. It should be noted, that a single injection of cartilage

proteoglycan does not induce arthritis.

To further investigate whether the T cell epitope represented by p135H might be

directly involved in the pathogenesis of the disease, we transferred p135H-stimulated

lymphocytes from peptide p135H-primed BALB/c mice into SCID mice. The clinical

appearance and histopathologic features of adoptively transferred arthritis in SCID mice

showed many similarities to PGIA, such as lymphocyte/lymphoblast infiltration in the

synovial membrane, pannus formation, synovial hyperplasia, and erosion of articular

cartilage and subcortical bone [5, 7]. According to the expression of cell surface

markers and the antigen (p135H)-induced cytokine profile, most of the peptide p135H-

specific lymphocytes were Th1 cells. These T cells cross-reacted in vitro with the

human, bovine, canine and porcine, as well as with the corresponding mouse (self),

aggrecan sequence. This cross-reactivity between the human and mouse sequences

74

indicated a high degree of epitope spreading that could establish the immunopathologic

basis of arthritis in BALB/c mice.

To further characterize T cells with p135H and p135M specificity, we have generated

peptide p135H-specific T cell hybridomas and used them to determine the fine epitope

specificity of TCR- and MHC-binding sites. Interestingly, while p135H- or p135M-

primed lymphocytes cross-reacted well with p135 peptides of the other species (Figure

3; section IV.1.2., page 46), only a few p135H-specific hybridomas were able to

transfer arthritis into “presensitized” SCID mice, whereas others, especially those

derived from p135M (mouse)-specific T cells, were unable to induce inflammation.

This might be explained by the different cytokine profile after peptide stimulation

and/or different epitope specificity of the T cell hybridomas (Figure 4; section IV.1.3.,

page 47 and Figure 8; section IV.1.6., page 53). Thus, while the sequence of p135

peptide allows for cross-reactivity between human- and mouse-specific T cell

populations, the epitope specificity of individual hybridomas is highly restricted to a

single peptide sequence. Essentially, all p135H-specific hybridomas generated in this

study recognized the same core sequence and TCR binding sites.

A special feature of this T cell epitope is that the p135H contains a conserved amino

acid sequence QKRSS, which shows striking similarity to the “shared epitope”

QKRAA. This “shared epitope”, and analogous sequences with conservative

replacements (QK/RRA/SA/S) are overrepresented in protein databases, suggesting that

this sequence motif may have a fairly ubiquitous function. Previous studies have shown

that this 5-amino acid motif is present in the third hypervariable region of certain HLA

alleles associated with RA (e.g. HLA–DR4/Dw4 and DR9) [12], and that the presence of

this “shared epitope” could predict a progressive destructive disease course [129]. HLA-

derived peptides encompassing the “shared epitope” sequence can randomly select T

75

cells that bind the self-derived peptide at low avidity. Later in life, these previously

quiescent QKRAA-specific T cells can be activated by binding with high avidity an

exogenous peptide containing the “shared epitope” [13-15].

Furthermore, it has been recently demonstrated that the “shared epitope” acts as a potent

immune-stimulatory ligand that activates a nitric oxide-mediated pathway in other cells

(e.g. dendritic cells), and can polarize T cell differentiation toward Th17 cells, a T cell

subset that has been implicated in the pathogenesis of autoimmune diseases, including

RA (Figure 2; section I.7., page 23)[112].

Interestingly, proteins in common human pathogens, such as Escherichia coli (DnaJ

heat-shock protein), Lactobacillus lactis and Brucella ovis (DnaJ heat-shock protein),

and Epstein-Barr virus (gp110 protein), have been identified that express the “shared

epitope” in the context of highly immunogenic proteins, and immune responses to

several of these antigens have been evaluated in RA patients [13-18]. In addition,

database searches reveal that peptide p135H also has a high sequence similarity (76%;

TTYKRR—RSSR) to the capsid protein VP3 of human JC polyomavirus, which is

ubiquitous in the human population and is the etiologic agent of progressive multifocal

leukoencephalopathy. The virus can infect children without producing symptoms; it

remains latent in the kidneys, tonsils and central nervous system, and may reactivate,

causing significant T cell dysfunction [130]. These sequence data suggest that

immunologic cross-reactivity might exist between certain RA-associated HLA alleles,

the above-mentioned immunogenic proteins of human pathogens, and p135H, a T cell

epitope of human cartilage proteoglycan. Indeed, a high cross-reactivity was observed

between peptide p135H and p135H-AA (Figure 3; section IV.1.2., page 46), a modified

version containing the “shared epitope” (Table 2; section III.2., page 31).

Intermittent exposure of the immune system to these bacterial or viral antigens at

76

mucosal surfaces might lead to the activation of resting QKRAA-specific T cells, and

then the T cell activation is perpetuated by encounters with peptides of self origin,

encompassing the “shared epitope” sequence. Why these lymphocytes show a pattern of

migration to the peripheral joints remains an open question, and the target of the

pathogenic autoimmune process in the joint is still elusive. It seems to be an attractive

hypothesis, however, that the sequence of p135H in human cartilage proteoglycan can

serve as a homolog of the “shared epitope”, and it may be responsible for the joint-

specific homing of QKRAA-reactive T lymphocytes and then be involved in the

initiation of the autoimmune process in RA (see Figure 15 below).

Loss of aggrecan is a major feature of cartilage degradation associated with arthritis

[27]. There is an age-related loss of the G3 domain of aggrecan, and 92% of the G3

domain is lost as part of the normal turnover of the proteoglycans [19], whereas the rest

of the molecule, which is bound to hyaluronan, is retained in the cartilage. The C-

terminal tail of aggrecan is cleaved first, and 2 tandem boxes (RRXXK and RXXR) of

the consensus sequences have been demonstrated to be involved in the earliest cleavage

[131]. Interestingly, both cleavage sites described are located within peptide p135H

(TTYKRRXXKRXXRHP). Therefore, a significant amount of p135H might be released

from articular cartilage due to normal turnover or due to an enhanced proteolytic

processing of the G3 domain (e.g. in inflammation or in cartilage injury), and then be

exposed to the immune system in the joints (Figure 15).

Since peptide p135H possesses a high sequence similarity to the “shared epitope”, it can

provide ground for “molecular mimicry”. Peptide p135H of the G3 domain can be

recognized by QKRAA-specific autoreactive T cells (Figure 3; section IV.1.2., page

46), which by migrating into the joint and expanding locally, might contribute to the

development of arthritis (Figure 5A-B; section IV.1.4., page 49). Therefore, we

77

suggest, that this C-terminal part of the G3 domain plays a role in the initial activation

of T cells, then later on the rest of the molecule, including the G1 domain becomes the

main target. This process might be interpreted as “epitope spreading”, that is in the

course of autoimmune inflammation neo-epitopes are generated, e.g. by citrullination of

other parts of the molecule, that subsequently become additional targets for the

autoimmune response, or by the enhanced proteolytic activity, exposing other (remnant)

epitopes to the immune system [30, 132, 133] (Figure 15).

In this study, we made efforts to further examine the role of peptide p135H, a T cell

epitope in the C-terminal G3 domain of human proteoglycan, in an experimental animal

model. Despite differences in the amino acid sequences between the human and mouse

peptide and the cryptic character of the epitope, we were able to show that the transfer

of peptide p135H-reactive lymphocytes into “presensitized” SCID mice leads to a rapid

development of arthritis. Hence, we have direct evidence that peptide p135H contains

an arthritogenic epitope that is recognized by T cells in mice and that it attracts these

cells to joints that contain the homologous mouse sequence, thus leading to induction of

arthritis.

Before, it has been assumed that all arthritogenic epitopes are located within the G1

domain of aggrecan. Purified G1 domain alone [10, 134] and G1 epitope-specific T cell

lines [135, 136] were capable of inducing arthritis when injected into BALB/c mice.

More importantly, patients with RA express cellular and humoral immunity to the G1

domain of cartilage proteoglycan aggrecan [137, 138]. To the best of our knowledge,

this is the first report of the induction of arthritis by T cells specific for an epitope in the

G3 domain of human cartilage proteoglycan.

78

Figure 15. The hypothetic role of p135H and DDA in the pathogenesis of rheumatoid

arthritis.

See discussion for a detailed description. Abbreviations used in the figure: ACPA anti-citrullinated

protein/peptide antibody, APC antigen presenting cell, CS chondroitin sulphate, DDA

dimethyldioctadecylammonium bromide, G1, 2, 3 globular domains 1, 2, 3 of aggrecan, IFN interferon,

IL interleukin, KS keratan sulphate, MHC major histocompatibility complex, OA osteoarthritis, p135H

human peptide p135, RA rheumatoid arthritis, RF rheumatoid factor, TCR T cell receptor, TNF tumor

necrosis factor.

In the second half of our studies, we have studied the adjuvant effect of

dimethyldioctadecylammonium bromide (DDA) on arthritis induction.

DDA belongs to the group of lipophilic quaternary amines that were described more

than 35 years ago as a potential adjuvant [95]. DDA as an adjuvant has been used

successfully and without side effects in vaccines administered to children and pregnant

women [96, 97]. It is also widely used as a detergent in cosmetic compounds and fabric

softeners [98]. DDA is a highly potent immunostimulator, especially with negatively

79

charged antigens, provoking strong delayed-type hypersensitivity [98, 100-102]. It is a

powerful, nonirritant adjuvant and, via T cell stimulation, significantly enhances

antigen-specific B cell activation and immunoglobulin production [103, 104]. A special

benefit of the use of DDA in rodent models of autoimmunity is that this adjuvant forces

the immunoregulation toward Th1 direction [105-107]. DDA can enhance the adjuvant

effect of other adjuvants [92] and even potentiate the arthritogenic effect of IFA [92] or

CFA [108] in oil adjuvant-susceptible strains of rats. However, DDA as an adjuvant has

never previously been tested in any autoimmune model of rheumatoid arthritis.

There are a number of inducible animal models of human autoimmune diseases, all of

which require immunization of genetically susceptible strains of rodents and non-human

primates with a target organ-specific antigen in adjuvant [93, 139, 140]. Depending on

the model and species, 1-4 injections of antigen are required, and at least 1 of these

injections should be given with CFA as adjuvant [93]. Therefore, CFA seems to be a

critical component in the induction and achievement of a high incidence and severity of

autoimmune diseases in rodent models. The use of the same (auto)antigen in IFA,

Alhydrogel (aluminum hydroxide gel) or synthetic adjuvants is either insufficient to

induce the disease, or the incidence and severity are far below those of the

antigen/CFA-induced disease. Thus, the nonspecific activation of innate immunity (e.g.

macrophages, dendritic cells) and T cells by mycobacterial components such as

muramyldipeptide (peptidoglycan), HSP and trehalosedimycolate (a glycolipid

equivalent with lipopolysaccharide of Escherichia coli) in mineral oil [92, 93] is a

critical component in the provocation of immune reactions to self antigens in

autoimmune models.

While a large number of adjuvants can be used for immunization of animals or for

vaccination of humans, until now CFA has remained the most potent and powerful

80

adjuvant in all experimental models of autoimmunity. The use of CFA, however,

induces several side effects, including immune reactions to mycobacterial components,

and as a highly irritating compound, CFA induces sterile inflammation, followed by

local granulomatous tissue formation and severe adhesions, especially in the peritoneal

cavity [93].

Here, we have compared the effects of DDA with those of CFA and IFA in arthritis-

susceptible murine strains to gain insight as to how the DDA could achieve an even

more superior effect than the Freund's adjuvants.

Among the adjuvants tested to date, only CFA and DDA proved to express sufficient

power to provoke arthritis in PG- or CII-immunized genetically susceptible strains of

mice. In terms of humoral and cellular immune responses to PG or other antigens and

production of related cytokines, CFA proved to be an equivalent, or an even more

effective adjuvant than DDA (Table 3; section IV.2.5., page 66 and Table 4; section

IV.2.5., page 67), and both CFA and DDA induced significantly higher immune

responses and cytokine production than did PG/IFA. However, the production of

antigen-specific Th1 and Th2 cytokines, and the shift of the Th1/Th2 balance toward

Th1 type response were significantly more pronounced in animals immunized with

PG/DDA, than in those immunized with PG/Freund's adjuvants (Table 3). Moreover, at

least a 2-4-fold increase in macrophage influx into the peritoneal cavity, accompanied

by more CD11c+ dendritic cells was characteristically observed in DDA-injected mice.

Furthermore, suboptimal doses of cartilage PGs or PGs having only a suboptimal

arthritogenic effect when injected with CFA, were as effective as the human cartilage

PGs in provoking PGIA, and crude extracts obtained from osteoarthritic cartilage, when

appropriately deglycosylated, induced arthritis in a manner similar to that of the highly

purified human cartilage PG, when DDA was used as adjuvant [141].

81

As a result, the use of DDA accelerated the development of a more severe arthritis, and

suboptimum doses of PG or CII antigens were able to induce inflammation via a more

potent activation of the innate immunity [141].

In RA there is abundant evidence that the innate immune system is persistently

activated, as evidenced by the continual expression of macrophage derived cytokines

such as TNF-α, IL-1 and IL-6. Furthermore, the central role of macrophages in RA

pathogenesis is supported by the fact that conventional therapies, including

methotrexate and cytokine inhibitors, act to decrease the production of cytokines

produced primarily by macrophages [142]. A potential or critical role of innate

immunity in arthritis induction is also consistent with the findings of studies that used

only adjuvants (nonspecific stimulators) in genetically susceptible strains of rodents [92,

93]. Observations from these studies of oil-, pristane- and squalene-induced arthritis

[92, 93, 143] together with our observations raise the question of whether the

“adjuvants” can also play a role in, and/or contribute to, joint inflammation in

genetically susceptible humans. Can immunostimulatory molecules from microbes,

environmental compounds (cosmetics, laundry detergents or food additives), or

endogenous “self” adjuvants (such as lipid squalene) in fact cause or contribute to joint

inflammation? Since a number of potential autoantigens (type II collagen, proteoglycan

aggrecan, link protein, gp-39 or glucose-phosphate isomerase) could be identified in

various subsets of RA patients, it may be an attractive hypothesis that nonspecific

stimulation of innate immunity might be an initial component in the disease mechanism.

A defect in immune regulation, the production of cytokines/chemokines, and the

possible involvement of joint-derived or joint-independent autoantigens (such as

rheumatoid factor) may be only subsequent, although clearly detectable, events in an

initial nonspecific activation of innate immunity. While the relevance of this

82

hypothesis requires extensive studies, our observations using an innocuous component

as adjuvant seem to support this possibility (Figure 15).

By now many supposed autoantigens have been described, and their finding was on the

search for the one and only autoreactive event that causes RA. However, it should be

noted, that none of the known RA-associated autoreactive events is present in all RA

patients, and none of them occurs exclusively in RA. In RA “self” is either the primary

target, or just a bystander that then becomes the focus of immune attack. The shift from

the normal state to RA involves autoreactive T and B cells directed at various synovial

and/or systemic antigens, activated antigen presenting cells, secretion of inflammatory

cytokines and autoantibodies, cartilage erosion and bone destruction, genetic

components such as the association with the „shared epitope”, and environmental

factors (e.g. infection). This complex network is then sufficient to maintain a self-

sustaining inflammation/arthritis, even without the permanent presence of the triggering

antigen.

According to our hypothesis, the two main subjects investigated in our studies, on the

one hand peptide p135H, representing a supposed autoantigen in the G3 domain of

human cartilage proteoglycan, and on the other hand DDA, a potent activator of innate

immunity, might be two players among others, contributing to the initiation,

perpetuation and joint-specificity of rheumatoid arthritis.

83

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VIII. BIBLIOGRAPHY

Cumulative impact factor: 35.973

PAPERS RELATED TO THIS THESIS (Impact factor: 17.26)

1. Hanyecz A, Bardos T, Berlo SE, Buzas E, Nesterovitch AB, Mikecz K, Glant TT.:

Induction of arthritis in SCID mice by T cells specific for the "shared epitope" sequence

in the G3 domain of human cartilage proteoglycan, Arthritis and Rheumatism, 2003,

48:2959-73, IF:7.19

2. Hanyecz A, Berlo SE, Szanto S, Broeren CP, Mikecz K, Glant TT.: Achievement of

a synergistic adjuvant effect on arthritis induction by activation of innate immunity and

forcing the immune response toward the Th1 phenotype, Arthritis and Rheumatism,

2004, 50(5):1665-76, IF: 7.19

3. Hanyecz A, Olasz K, Tarjanyi O, Nemeth P, Mikecz K, Glant TT, Boldizsar F.:

Proteoglycan aggrecan conducting T cell activation and apoptosis in a murine model of

rheumatoid arthritis, BioMed Research International, 2013, accepted for publication, IF:

2.88

OTHER PAPERS PUBLISHED IN PERIODICALS (Impact factor: 18.713)

1. Buzas EI, Hanyecz A, Murad Y, Hudecz F, Rajnavolgyi E, Mikecz K, Glant TT.:

Differential recognition of altered peptide ligands distinguishes two functionally

discordant (arthritogenic and nonarthritogenic) autoreactive T cell hybridoma clones,

Journal of Immunology, 2003, 171:3025-33, IF: 7.014

2. Firneisz G, Zehavi I, Vermes C, Hanyecz A, Frieman JA, Glant TT.: Identification

96

and quantification of disease-related gene clusters, Bioinformatics, 2003, 19:1781-6, IF:

6.701

3. Adarichev VA, Vermes C, Hanyecz A, Mikecz K, Bremer EG, Glant TT.: Gene

expression profiling in murine autoimmune arthritis during the initiation and

progression of joint inflammation., Arthritis Research and Therapy, 2005, 7(2):R196-

207, IF: 2.965

4. Adarichev VA, Vermes C, Hanyecz A, Ludanyi K, Tunyogi-Csapo M, Finnegan A,

Mikecz K, Glant TT.: Antigen-induced differential gene expression profile in synovium

prior to the onset of arthritis, Autoimmunity, 2006, 39(8):663-673, IF: 2.033

PEER REVIEWED AND PUBLISHED ABSTRACTS

1. Vermes, C., Roebuck, K.A., Fritz, E.A., Hanyecz, A., Glant, T.T.: Shedding of non-

functional interleukin-6 (IL-6) receptor (IL6R or gp80) results in the activation of

gp130-mediated signaling in human osteoblasts, Arthritis and Rheumatism, 2001, 44:

S360

2. Czipri, M., Bardos, T., Stoop, R., Vermes, C., Gal, I., Hanyecz, A., Mikecz, K.,

Watanabe, H., Yamada, Y., Glant, T.T.: A novel approach for gene therapy: complete

rescue of otherwise embryonic lethal defect in skeletal development, Arthritis and

Rheumatism, 2001, 44: S581

3. Firneisz, G., Hanyecz, A., Vermes, C., Bardos, T., Glant, T.T.: Gene expression

profile in an animal model of rheumatoid arthritis using cDNA microarrays, Arthritis

and Rheumatism, 2001, 44: S747

4. Bardos, T., Hanyecz, A., Chella, S.D., Glant, T.T.: Human MHC class II transgenic

mice recognize peptide epitopes of human cartilage proteoglycan (PG), Arthritis and

Rheumatism, 2001, 44: S1473

97

5. Vermes, C., Bardos, T., Czipri, M., Hanyecz, A., Lovasz, G., Bellyei, A., Fritz, E.A.,

Roebuck, K.A., Jacobs, J.J., Galante, J.O., Andersson, G.B.J., Glant, T.T.: Differential

effects of bacterial lipopolysacharide (LPS) and tumor necrosis factor-alpha on the

functions of human osteoblast cells, Orthop. Trans., 2002, 27:537

6. Czipri, M., Bardos, T., Vermes, C., Hanyecz, A., Gal, I., Mikecz, K., Watanabe, H.,

Yamada, Y., Glant, T.T.: Genetic rescue of an otherwise perinatal lethal defect in

skeletal development, Orthop. Trans., 2002, 27:311

7. Hanyecz, A., Bardos, T., Berlo, S.E., Mikecz, K., Glant, T.T.: A T cell hybridoma,

specific for an epitope in the G3 domain of human cartilage proteoglycan, induces

arthritis in SCID mice, Arthritis and Rheumatism, 2002, 46:S300

8. Hanyecz, A., Bardos, T., Berlo, S.E., Mikecz, K., Vermes, C., Glant, T.T.: A novel

approach creating a "prearthritic" stage, for investigating the in vivo arthritogenic effect

of T cell hybridomas, Arthritis and Rheumatism, 2002, 46:S483

9. Szanto, S., Bardos, T., Hanyecz, A., Chella, D.S., Glant, T.T.: Human MHC class II

transgenic mice (HLA-DR4 and HLA-DQ8) recognize peptide epitopes of human

cartilage proteoglycan (PG), but develop arthritis only in a genetically susceptible

background, Arthritis and Rheumatism, 2003, 48:S148-S149

10. Firneisz, G., Zehavi, I., Vermes, C., Hanyecz, A., Frieman, J.A., Glant, T.T.: A

novel combination of methods to identify and quantify disease-related gene clusters,

Arthritis and Rheumatism, 2003, 47:S447

11. Adarichev, V.A, Vermes, C, Hanyecz, A, Bremer, E.G, Glant, T.T.: Profiling gene

expression in antigen-stimulated lymphocytes, cells which induce arthritis in syngeneic

SCID mice, Arthritis and Rheumatism, 2004, 50:S123

12. Adarichev, V.A, Vermes, C, Hanyecz, A, Bremer, E, Glant T.T.: Gene expression

profiling in murine adoptively transferred arthritis: Interaction of innate and adaptive

98

immunity, Arthritis and Rheumatism, 2004, 50:S122-S123

BOOK CHAPTER

Buzás Edit, Hanyecz Anita, Szekanecz Zoltán: Animal models of arthritis, Clinical

Immunology, edited by Prof. Dr. Czirják László, published by Medicina, Budapest,

2006, 2nd chapter: 101-105.

PODIUM AND POSTER PRESENTATIONS

1. Bardos, T., Hanyecz, A., Buzas, E.I., Glant T.T.: Pre- and co-immunization with self

and non-self peptide sequences significantly modify proteoglycan-induced arthritis in

BALB/c mice

Rush University Forum for Research and Clinical Investigation, 2001, Chicago, Illinois,

USA

2. Vermes, C., Roebuck, K.A., Fritz, E.A., Hanyecz, A., Glant, T.T.: Shedding of non-

functional interleukin-6 (IL-6) receptor (IL6R or gp80) results in the activation of

gp130-mediated signaling in human osteoblasts

65th

Annual Scientific Meeting, American College of Rheumatology, 2001, San

Francisco, California, USA

3. Czipri, M., Bardos, T., Stoop, R., Vermes, C., Gal, I., Hanyecz, A., Mikecz, K.,

Watanabe, H., Yamada, Y., Glant, T.T.: A novel approach for gene therapy: complete

rescue of otherwise embryonic lethal defect in skeletal development

65th

Annual Scientific Meeting, American College of Rheumatology, 2001, San

Francisco, California, USA

4. Firneisz, G., Hanyecz, A., Vermes, C., Bardos, T., Glant, T.T.: Gene expression

profile in an animal model of rheumatoid arthritis using cDNA microarrays

99

65th

Annual Scientific Meeting, American College of Rheumatology, 2001, San

Francisco, California, USA

5. Bardos, T., Hanyecz, A., Chella, S.D., Glant, T.T.: Human MHC class II transgenic

mice recognize peptide epitopes of human cartilage proteoglycan (PG)

65th

Annual Scientific Meeting, American College of Rheumatology, 2001, San

Francisco, California, USA

6. Vermes, C., Bardos, T., Czipri, M., Hanyecz, A., Lovasz, G., Bellyei, A., Fritz, E.A.,

Roebuck, K.A., Jacobs, J.J., Galante, J.O., Andersson, G.B.J., Glant, T.T.: Differential

effects of bacterial lipopolysacharide (LPS) and tumor necrosis factor-alpha on the

functions of human osteoblast cells

48th

Annual Meeting, Orthopedic Research Society, 2002, Dallas, Texas, USA

7. Czipri, M., Bardos, T., Vermes, C., Hanyecz, A., Gal, I., Mikecz, K., Watanabe, H.,

Yamada, Y., Glant, T.T.: Genetic rescue of an otherwise perinatal lethal defect in

skeletal development ("New Investigator Recognition Award"-winning poster)

48th

Annual Meeting, Orthopedic Research Society, 2002, Dallas, Texas, USA

8. Firneisz, G., Hanyecz, A., Vermes, C., Bardos, T., Glant, T.T.: Gene expression

profile in an animal model of rheumatoid arthritis using cDNA microarrays

Rush University Forum for Research and Clinical Investigation, 2002, Chicago, Illinois,

USA

9. Vermes, C., Hanyecz, A., Andersson, G., Jacobs, J.J., Roebuck, K.A., Glant, T.T.:

Shedding of the IL-6 receptor determines the ability of IL-6 to induce GP130-

phosphorylation in human osteoblasts

Rush University Forum for Research and Clinical Investigation, 2002, Chicago, Illinois,

USA

10. Vermes, C., Hanyecz, A., Raman, C., Andersson, G., An, H., Jacobs, J.J., Dobai,

100

J., Roebuck, K.A., Glant, T.T.: Pamidronate and 1,25-dihydroxy-vitamin-D3 inhibit

tumor necrosis factor-alpha- and wear debris-induced interleukin-6 release and recover

suppressed type 1 collagen synthesis in bone marrow-derived primary human

osteoblasts

Rush University Forum for Research and Clinical Investigation, 2002, Chicago, Illinois,

USA

11. Hanyecz, A., Bardos, T., Berlo, S.E., Mikecz, K., Glant, T.T.: A T cell hybridoma,

specific for an epitope in the G3 domain of human cartilage proteoglycan, induces

arthritis in SCID mice

66th

Annual Scientific Meeting, American College of Rheumatology, 2002, New

Orleans, Louisiana, USA

12. Hanyecz, A., Bardos, T., Berlo, S.E., Mikecz, K., Vermes, C., Glant, T.T.: A novel

approach creating a "prearthritic" stage, for investigating the in vivo arthritogenic effect

of T cell hybridomas

66th

Annual Scientific Meeting, American College of Rheumatology, 2002, New

Orleans, Louisiana, USA

13. Berlo, S.E., Hanyecz, A., van Kooten, P., ten Brink, C., Glant, T.T., Prakken, B.,

van der Zee, R., Singh, M., van Eden, W., Broeren, C.: Immune modulation in

proteoglycan-induced arthritis

"Translational research in autoimmunity" meeting, 2003, Genova, Italy

14. Szanto, S., Bardos, T., Hanyecz, A.,. Chella, D.S., Glant, T.T.: Human MHC class

II transgenic mice (HLA-DR4 and HLA-DQ8) recognize peptide epitopes of human

cartilage proteoglycan (PG), but develop arthritis only in a genetically susceptible

background

67th

Annual Scientific Meeting, American College of Rheumatology, 2003, San

101

Francisco, California, USA

15. Firneisz, G., Zehavi, I., Vermes, C., Hanyecz, A., Frieman, J.A., Glant, T.T.: A

novel combination of methods to identify and quantify disease-related gene clusters

67th

Annual Scientific Meeting, American College of Rheumatology, 2003, San

Francisco, California, USA

16. Hanyecz A.: Role of the “shared epitope” in the development of rheumatoid

arthritis, 2004, Annual Forum of Residents in Rheumatology, University of Pécs,

Faculty of Medicine, Department of Immunology and Rheumatology

17. Hanyecz A, Szász O., Moezzi M., Battyáni Z.: Severe cases of drug allergy, 2012,

Pécs, Dermatology and its bounderies – 1st multidisciplinar course, University of Pécs,

Faculty of Medicine, Department of Dermatology, Venereology and Dermatooncology

102

IX. ACKNOWLEDGEMENTS

Herein I would like to thank my mentor Dr. Ferenc Boldizsár, and Ph.D. program leader

Prof. Péter Németh for giving me the possibility to complete my Ph.D. work at the

Department of Immunology and Biotechnology, and all the support and scientific

guidance they gave me for the preparation of the thesis.

I would like to express my deepest appreciation to my honorary mentor Prof. Tibor T.

Glant for giving me the possibility to work for three years at Rush University (Chicago,

IL, USA), and for introducing me the proteoglycan-induced experimental arthritis

model, teaching me several modern laboratory technics, and for his guidance and

encouragement in this scientific endeavor during these years.

I acknowledge the contributions of many colleagues in the Department of Molecular

Medicine at Rush University Medical Center to this work: Prof. Katalin Mikecz, Prof.

Alison Finnegan, Dr. Tamás Bárdos, Dr. Csaba Vermes, Suzanne Berlo, Dr. Sándor

Szántó, Sonja Velins and Kevin Kolman among others.

Especially, I express my most sincere thank and gratitude to my family, particularly my

parents, my husband and my children for their love and encouraging support.

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X. APPENDIX