| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Department of Immunology and Bacteriology, University of Glasgow, Western Infirmary, Glasgow, United Kingdom; and
Center for Rheumatic Diseases, University Department of Medicine, Glasgow Royal Infirmary, Alexandra Parade, Glasgow, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Development of an arthritis model in which the precise contribution of DCs could be examined would therefore be of considerable value in understanding the pathogenesis and evaluating novel therapeutics. Ag-pulsed DCs have been shown to induce disease in a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (24). In contrast, the roles that DCs may play in the induction of experimental models of arthritis remain uncharacterized. Although collagen-induced arthritis (CIA) in DBA/1 mice has previously been used to estimate the relative contribution of immune cell subsets, significant problems arise in interpreting mechanisms whereby articular inflammation is induced, reflecting the ill-defined contribution of CFA (25, 26) which is necessary for CIA induction. Interestingly, IFA results only in the development of low-grade CIA, illustrating the essential role of bacteria or bacterial extracts for arthritis induction (27). One possible role of these bacterial products may be to activate DCs that are presenting arthritogenic peptides (18, 19). In the current study, we demonstrate for the first time that transfer of collagen-pulsed mature DCs is sufficient to induce arthritis in DBA/1 mice. Furthermore, we have used this novel model to elucidate that this disease process is mediated through two distinct effects of DCs, one involving priming of autoreactive T cells and a second related to the induction of proinflammatory agents such as TNF.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DCs were generated from the bone marrows of DBA/1 mice (obtained
from Harlan Olac, Bicester, U.K.) by culture in GM-CSF-conditioned
medium as previously described (28). FACS analysis of
CD11c and MHC class II (I-Aq; BD Biosciences, Oxford, U.K.)
expression confirmed routine yields of >80% pure DC cultures, with no
detectable levels of expression of CD8
(BD Biosciences). DCs were
activated by overnight incubation with 1 µg/ml LPS (from
Salmonella abortus equi; Sigma-Aldrich, Poole, U.K.) and,
where appropriate, were simultaneously loaded with type II collagen (50
µg/ml; Sigma-Aldrich) or OVA (100 µg/ml; Worthington Biochemical,
Lakewood, NJ). Overnight incubation of these cells with LPS, with or
without Ag, produced mature DCs characterized by increased
surface expression of class II MHC, CD40, CD80, and CD86
(28). Prior to transfer, cells were collected and washed
with RPMI 1640 supplemented with 100 IU/ml penicillin, 100 µg/ml
streptomycin, and 2 mM L-glutamine (incomplete RPMI
1640; all from Life Technologies, Paisley, U.K.).
Induction and assessment of DCIA
Arthritis was induced in male DBA/1 mice (Harlan Olac) by s.c. administration of 2 x 105 collagen-pulsed mature DCs into each hind footpad. Control mice received mature DCs pulsed with OVA or mature unpulsed DCs. Mice were subsequently monitored for signs of arthritis as described previously (27). Disease was scored based on erythema, swelling, or loss of function present in each paw on a scale of 0–3, giving a maximum score of 6 per mouse. Paw thickness was measured with a dial caliper (Kroeplin, Munich, Germany). The effects of TNF neutralization on the development of DC-induced arthritis (DCIA) was determined by i.p. administration of TNF receptor type II-IgG fusion protein (Wyeth-Ayerst Pharmaceuticals, St. Davids, PA), or control IgG (50 µg/mouse) every 2 days from the onset of disease. In some experiments, T cells were purified from peripheral lymph nodes of DC-transferred mice using T cell enrichment columns (Biotex Laboratories, Edmonton, Canada). This method produced cell suspensions containing >95% CD3+ T cells as assessed by FACS analysis. Purified T cells (1 x 106) were then transferred i.v. into DBA/1 mice and the development of arthritis was monitored as described above. To determine the effects of inflammation and lymphocyte recruitment on the spread of disease, TNF, or the CXCR4 chemokine, stromal cell-derived factor 1 (SDF-1; Ref. 29) (50 ng/mouse; both from R&D Systems, Oxon, U.K.) was injected s.c. into the hind footpads of DBA/1 mice on days 20 and 21 following priming with collagen-pulsed DCs. In T cell transfer studies, TNF (50 ng/mouse) was injected into the hind footpads of mice on days 0 and 1 following T cell transfer. Control mice received s.c. PBS only. For histological assessment, mice were sacrificed and the hind limbs were removed and fixed in 10% neutral-buffered Formalin, and 5-µm sections were stained with H&E or toludine blue (both from Sigma-Aldrich). The quantification of arthritis was by "treatment-blind" observers and a histological score was assigned to each joint based on the degree of inflammation, synovial hyperplasia, and erosion as described previously (30).
Fluorescence microscopy
For fluorescence imaging, DCs were labeled by incubating in incomplete RPMI 1640 containing 1 µM CellTracker Green 5-chloromethyl-fluorescein diacetate (CMFDA; Molecular Probes, Eugene, OR) for 30 min at 37°C (31). Cells were then washed and resuspended in complete RPMI 1640 (plus 10% FCS; Life Technologies, Grand Island, NY) for 30 min to activate the fluorescent probe. Cells were washed and resuspended in incomplete medium immediately prior to transfer.
Draining lymph nodes were removed 48 and 96 h following transfer and snap frozen in liquid nitrogen. Sections (6–8 µm) were cut from these tissues, fixed in acetone, and treated with 0.1% sodium azide/3% H2O2 in PBS to quench endogenous peroxidase. Fc receptor binding of Abs was blocked with anti-CD16/CD32 Ab (2.4G2; BD Biosciences) prepared in 10% mouse serum and 0.05% azide. Sections were then labeled with PE anti-CD4 (L3T4; BD Biosciences) or rabbit antiproliferating cell nuclear Ag (PCNA; Santa Cruz Biotechnology, Santa Cruz, CA) (32). PCNA labeling was amplified using biotin anti-rabbit IgG (Vector Laboratories, Burlingame, CA) and streptavidin HRP (Vector Laboratories) followed by the tyramide signal amplification biotin system (NEN Life Science Products, Boston, MA). PCNA staining was visualized using Texas Red-streptavidin (Vector Laboratories). Control and labeled tissue sections were examined on an Axioskop microscope (Zeiss, Oberkochen, Germany) and images were captured by a cooled charge-coupled device camera (Digital Pixel, Brighton, U.K.) onto an Apple Macintosh computer using IP lab software (Scanalytics, Fairfax, VA). Images were then processed using Adobe Photoshop version 5.0 (Adobe Systems, San Jose, CA).
Restimulation of draining lymph node cells in vitro
Draining lymph nodes were removed at the end of in vivo experiments. Single-cell suspensions were prepared and cultured in triplicate at 2 x 106 cells/ml in RPMI 1640 containing 1% mouse serum at 37°C in 5% CO2. Cells were stimulated with either medium alone, 50 µg/ml type II collagen, or 100 µg/ml OVA in flat-bottom 96-well plates (Nunc, Roskilde, Denmark). Supernatants were harvested after 72 h and stored at -20°C for cytokine assay. Proliferation assays were performed in parallel cultures in U-bottom 96-well plates (Nunc) for 96 h and were pulsed with [3H]thymidine (Amersham, Aylesbury, U.K.) during the last 6 h of culture. Cells were then harvested and measured for incorporation of radioactivity as previously described (27).
Cytokine ELISA
All cytokines were detected by ELISA as described previously
(27). The Ab pairs were as follows: TNF (BioSource
International, Nivelles, Belgium), IFN-
(R&D Systems), IL-2, IL-5,
IL-6, and IL-10 (BD Biosciences), and assays were performed according
to the manufacturers’ instructions. Detection limits were as follows:
IL-2, IL-5, IL-6, and TNF all at 10 pg/ml; IL-10 at 20 pg/ml and
IFN- at 40 pg/ml.
Statistical analysis
Statistical analysis was performed using Minitab software (Minitab, State College, PA). Clinical and histological scores were analyzed with the nonparametric Mann-Whitney U test. Cytokine levels were compared using Student’s t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mature DCs were transferred s.c. into the footpads of DBA/1 mice
and the subsequent development of inflammatory arthritis was monitored
for frequency (Fig. 1
A) and
severity (mean articular index; Fig. 1B). Transfer of
collagen-pulsed mature DCs into recipient mice was sufficient for the
induction of arthritis, first evident at day 10 after cell transfer in
up to 80% of recipients. Development of DCIA was partially inhibited
by TNFRp75-Ig but not control Ig administration (Fig. 1C).
Collagen-specific responses were critical since, in contrast, transfer
of mature, unpulsed DCs alone, or OVA-pulsed DCs did not induce
arthritis. Sequential histological analyses were performed which
revealed early predominance of neutrophils up to day 20 followed by
mononuclear cell infiltration. Established DCIA exhibited extensive
synovial hyperplasia and mononuclear
inflammatory cell infiltration (Fig. 2, A and C;
Table I). Appearances resembled those of
CFA-induced CIA (30) and RA synovial tissue (33, 34). Commensurate with clinical observations, joints of
mice receiving unpulsed DCs exhibited no apparent local inflammatory
reaction (Fig. 2, B and D).
|
|
|
We next characterized the nature of T cell responses in DC
recipients. When draining lymph node cells were restimulated with
collagen in vitro, significant levels of proliferation and cytokine
production were detected only in cultures prepared from collagen-pulsed
DC recipients (p < 0.05) and not unpulsed DC
recipients (Fig. 3). Ag-dependent
production of proinflammatory cytokines including TNF and IL-6
was detected (p < 0.05, Fig. 3, B–E). Similarly, significant IFN- and IL-2 production
was observed (p < 0.05), whereas IL-5 was not
detected, suggesting a predominant Th1-type response (35, 36). Spontaneous production of IL-10 could also be detected,
which was not however further induced by incubation with collagen in
vitro (Fig. 3F).
|
, A–D). To determine
whether DCs had any functional effect on local T cells, we analyzed the
expression of PCNA (32) (Fig. 4, E and
F). PCNA-positive cells could be identified adjacent to the
transferred DCs, indicating that local DNA synthesis and proliferation
were taking place in the T cell areas of the lymph node.
|
We next investigated whether the primary role of DCs in this model was to induce Ag-specific T cell responses and thereby initiate disease. We generated collagen- or OVA-specific T cell populations by injecting appropriately pulsed mature DCs into DBA/1 mice. T cell populations were then purified from lymph nodes of immunized mice and were transferred i.v. into congenic recipients. Ag-specific proliferation and cytokine production were observed in cultures of appropriately restimulated splenocytes isolated from these recipient mice 12 days after cell transfer (data not shown). However, arthritis onset was not observed in the majority of recipient mice (only one collagen-specific T cell recipient developed low-grade arthritis; data not shown). These data suggested that T cell priming alone was an insufficient explanation for the role of DCs in DCIA.
TNF regulates arthritis induction in collagen-pulsed DC recipients
An important feature of DCIA was the exclusive localization of
arthritis to those joints proximal to the site of DC transfer (Fig. 5, A and B) despite
the presence of recirculating collagen-reactive T cells. It seemed
likely that disease localization arose from signals delivered in the
articular environment by DCs themselves, irrespective of specific Ag
presentation. To test this hypothesis, we administered
noncollagen-pulsed mature DCs into the contralateral footpads of mice
originally primed with collagen-pulsed DCs. This resulted in
contralateral transfer of disease into the previously uninvolved
joints (Fig. 5C). Such inflammation arose with more rapid
kinetics than initial disease onset and was associated with recurrence
of left footpad inflammation representing bilateral disease. In
contrast, mice that initially received noncollagen-pulsed DCs and that
subsequently received noncollagen-pulsed DCs in the contralateral
footpads did not develop any signs of arthritis (data not shown).
|
D). Whereas sham injection with PBS
did not induce local inflammation, administration of TNF (Fig. 5D) resulted in induction of arthritis in joints local to
the site of injection in >70% of the mice. The induction of arthritis
in these joints also occurred rapidly, within 3–6 days following
treatment with TNF. This was not merely dependent on chemokinetic
activity since SDF-1 was ineffective in inducing local disease (data
not shown). In contrast, mice that initially received
noncollagen-pulsed DCs and that subsequently received TNF in the
contralateral footpad did not develop any signs of arthritis (data not
shown).
These data also predicted that TNF injection might be sufficient to
overcome the previously noted inability of adoptively transferred
collagen-specific T cells to induce arthritis. Thus, hind footpad
injection of TNF induced rapid onset of severe arthritis in 80% of
collagen-specific T cell recipients (Fig. 6). Local Ag recognition was crucial
since OVA-primed T cell recipients did not develop disease despite
local TNF injection (Fig. 6). Moreover, local TNF injection also
produced a significant increase in Ag-specific spleen cell
proliferation and cytokine production in mice that had received
collagen-specific T cells (p < 0.05).
Importantly, this was not the case when OVA-primed cells had been
transferred (Fig. 7). These data suggest
that arthritis induction was associated with enhanced systemic
responses to collagen but not to OVA in vivo.
|
|
). This
clearly indicated that one effect of TNF blockade in RA in vivo could
be mediated through suppression of DC-mediated amplification of
articular inflammation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Activation of DCs involves a number of phenotypic changes which are appropriate for enhanced T cell activation, as well as modulation of chemokine receptors resulting in the migration of DCs to the T cell areas of local lymph nodes (44, 45). In the present study, collagen-pulsed DCs (CMFDA+) could be observed in the T cell areas of popliteal and inguinal lymph nodes 2 days after transfer, and some of the CD4 cells associated with these DCs were activated (PCNA+) (32). These observations suggest that the role of DCs in the inductive phase of arthritis in this model involves activation of T cells in local lymph nodes.
An interesting feature of the model described here is the lack of induction of polyarthritis, a characteristic of other arthritic disease models (41, 42). Thus, upon transfer of DCs into the footpad of recipients, arthritis is only observed in the local joints and does not spread contralaterally or into any of the anterior joints despite clear evidence of a systemic, collagen-specific T cell response. Although no gross inflammation was evident at the site of injection, it is likely that the transient and focal inflammation induced by administration of DCs may be sufficient to induce local endothelial activation and to allow primed T cells access to the local joints. This hypothesis is supported by our observation that arthritis developed local to the site of injection of unpulsed DCs in mice previously primed in the contralateral footpad with collagen-pulsed, but not unpulsed DCs. These studies therefore demonstrate the importance not only of effective T cell priming but also of local inflammation in the induction of disease following DC transfer. Central to this event is TNF, which allows T cells to gain access to tissues through its ability to activate endothelial cells and to induce chemokine gradients (46).
To test this hypothesis, we administered low doses of TNF or chemokines (SDF-1) into the uninvolved footpads of mice that had already been primed by transfer of DCs in the contralateral footpads. Arthritis developed in the TNF-treated, but not the SDF-1-treated footpads of mice primed with collagen, but not OVA-pulsed DCs. Because SDF-1 failed to induce spread of disease into uninvolved joints, these results further emphasize the primary role played by endothelial activation in the induction of arthritis. Arthritis induced by injection of unpulsed DCs into the contralateral footpad could be partially inhibited by treatment with TNFRp75-Ig, indicating that DCs may produce TNF or act indirectly to induce TNF production in vivo. The lack of complete inhibition may be due to incomplete neutralization of TNF or the involvement of other mediators in addition to TNF in the activation of disease.
Interestingly, we also observed that TNF treatment of mice receiving collagen-primed T cells increased in vitro splenocyte responses, whereas administration of TNF to OVA-primed T cell recipients did not produce an equivalent increase in T cell reactivity on exposure to OVA in vitro. The collagen-specific effect of TNF suggests further roles for this cytokine, in addition to the induction of T cell egress, in arthritis development. It appears likely that following TNF treatment, collagen-specific T cells can gain access to the joint where they recognize autoantigen and become further activated. This secondary event may then allow the activation of synovial DCs, as observed previously (6, 7, 8), thus inducing further local activation of autoantigen-specific T cells. This function of TNF may be distinct from its pathological role associated with the inflammatory cytokine cascade. Commensurate with this, recent studies analyzing the clinical effects of anti-TNF therapy (infliximab) in patients with RA showed a decrease in the number of joint-infiltrating lymphocytes and a corresponding increase in circulating lymphocytes on initiation of treatment (47). Collectively, these data indicate two significant roles for TNF in arthritis, one during DC maturation and a second in activating the vascular endothelium to allow access of reactive T cells into the joint to initiate synovial inflammation.
Our results therefore not only demonstrate a novel model of experimental arthritis, but also reveal a dual role for DCs in the induction of arthritis. These studies have also significantly advanced our understanding of the mechanisms of anti-TNF therapy in RA.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. James M. Brewer, Department of Immunology and Bacteriology, University of Glasgow, Western Infirmary, Glasgow, G11 6NT, U.K. E-mail address: j.m.brewer{at}clinmed.gla.ac.uk
3 Abbreviations used in this paper: RA, rheumatoid arthritis; DC, dendritic cell; DCIA, DC-induced arthritis; CIA, collagen-induced arthritis; SDF-1, stromal cell-derived factor 1; CMFDA, 5-chloromethyl-fluorescein diacetate; PCNA, proliferating cell nuclear Ag.
Received for publication July 23, 2002. Accepted for publication October 18, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and the possible absence of specific immune reactions. Curr. Opin. Immunol. 11:657.[Medline]
-positive dendritic cells from a common myeloid progenitor. Science 290:2152. and transforming growth factor 
during induction of collagen type II arthritis in mice. Proc. Natl. Acad. Sci. USA 89:7375. anti-CD4, and combined anti-tumor necrosis factor /anti-CD4 treatment in early collagen-induced arthritis. Arthritis Rheum. 43:638.[Medline]
and interleukin-1 for migration. Immunology 92:388.[Medline]
This article has been cited by other articles:
|
|
 |
|
  A. L. Zhang, P. Colmenero, U. Purath, C. Teixeira de Matos, W. Hueber, L. Klareskog, I. H. Tarner, E. G. Engleman, and K. Soderstrom Natural killer cells trigger differentiation of monocytes into dendritic cells Blood, October 1, 2007; 110(7): 2484 - 2493. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Izawa, N. Ishimaru, K. Moriyama, M. Kohashi, R. Arakaki, and Y. Hayashi Crosstalk between RANKL and Fas signaling in dendritic cells controls immune tolerance Blood, July 1, 2007; 110(1): 242 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T R D J Radstake, K C A M Nabbe, M H Wenink, M F Roelofs, A Oosterlaar, A W T van Lieshout, P Barrera, P L E M van Lent, and W B van den Berg Dendritic cells from patients with rheumatoid arthritis lack the interleukin 13 mediated increase of Fc{gamma}RII expression, which has clear functional consequences Ann Rheum Dis, December 1, 2005; 64(12): 1737 - 1743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Moghaddami, L. G. Cleland, and G. Mayrhofer MHC II+ CD45+ cells from synovium-rich tissues of normal rats: phenotype, comparison with macrophage and dendritic cell lineages and differentiation into mature dendritic cells in vitro Int. Immunol., August 1, 2005; 17(8): 1103 - 1115. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yoshitomi, N. Sakaguchi, K. Kobayashi, G. D. Brown, T. Tagami, T. Sakihama, K. Hirota, S. Tanaka, T. Nomura, I. Miki, et al. A role for fungal {beta}-glucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice J. Exp. Med., March 21, 2005; 201(6): 949 - 960. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A W T van Lieshout, P Barrera, R L Smeets, G J Pesman, P L C M van Riel, W B van den Berg, and T R D J Radstake Inhibition of TNF{alpha} during maturation of dendritic cells results in the development of semi-mature cells: a potential mechanism for the beneficial effects of TNF{alpha} blockade in rheumatoid arthritis Ann Rheum Dis, March 1, 2005; 64(3): 408 - 414. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Maffia, J. M. Brewer, J. A. Gracie, A. Ianaro, B. P. Leung, P. J. Mitchell, K. M. Smith, I. B. McInnes, and P. Garside Inducing Experimental Arthritis and Breaking Self-Tolerance to Joint-Specific Antigens with Trackable, Ovalbumin-Specific T Cells J. Immunol., July 1, 2004; 173(1): 151 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T R D J Radstake, P L E M van Lent, G J Pesman, A B Blom, F G J Sweep, J Ronnelid, G J Adema, P Barrera, and W B van den Berg High production of proinflammatory and Th1 cytokines by dendritic cells from patients with rheumatoid arthritis, and down regulation upon Fc{gamma}R triggering Ann Rheum Dis, June 1, 2004; 63(6): 696 - 702. [Abstract] [Full Text]
|
|
|
|
 |
|
 L. S. Davis A Question of Transformation: The Synovial Fibroblast in Rheumatoid Arthritis Am. J. Pathol., May 1, 2003; 162(5): 1399 - 1402. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
