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,
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Departments of
*
Medicine and
Pediatrics, Division of Rheumatology, Allergy and Immunology, and Samana Rose Stein Institute for Research on Aging, University of California at San Diego, La Jolla, CA 92093; and
University Medical Center, Wilhelmina Children’s Hospital, Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and increased production
of the osteoclast differentiation factor, receptor activator of NF-
B
ligand (RANKL). The Mtb DNA did not enter the joints but dispersed to
the bone marrow and spleen before the onset of systemic joint
inflammation. Thus, adjuvant arthritis is a microbial DNA-dependent
disease. In this model, we postulate that massive and prolonged
activation of macrophages, dendritic cells, and osteoclast precursors
in the bone marrow may prime the joints for the induction of
inflammatory Th1 immune responses to Mtb Ags. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IFN by macrophages,
dendritic cells, B lymphocytes, and NK cells (1).
Consequent to this effect on innate immunity, ISS skew adaptive immune
responses toward a strong and prolonged Th1 type of immunity (3, 4).
Recently, Tarkowski and coworkers (5) were able to link
joint damage in a model of septic arthritis to the presence of ISS.
They showed that direct injection of bacterial DNA or oligonucleotides
containing ISS into the joints led to arthritis, whereas injection of
mammalian DNA did not induce inflammation. An influx of monocytes and
macrophages, and only a minority of CD4-positive T lymphocytes,
characterized the resulting arthritis. Locally, in the arthritic joint,
an increased expression of mRNA was found for TNF-, IL-1, and
IL-12, and for the chemokines RANTES and monocyte chemoattractant
protein-1 (6). Altogether, these results indicated that
bacterial DNA was sufficient to provoke joint inflammation in septic
arthritis, and raised the question as to whether ISS might also play a
role in the pathogenesis of other forms of arthritis (6).
To explore this issue, we turned to a T cell-mediated model of
experimental autoimmune arthritis, namely adjuvant arthritis (AA). AA
is an extensively studied form of chronic arthritis with a close
histopathological resemblance to rheumatoid arthritis (7, 8). AA can be induced in susceptible animals such as Lewis rats
by injection of heat-killed Mycobacterium tuberculosis (Mtb)
in IFA in the base of the tail. Because the immunostimulatory
properties of bacterial DNA were first discovered in Mtb
(9), we reasoned that ISS might play a role in this model
of autoimmune arthritis. In this study, we show that, indeed, ISS are
crucial for the induction of maximal joint inflammation in AA and
strongly stimulate both a Th1 response and the production of the
osteoclast differentiating factor, receptor activator of NF-B ligand
(RANKL), to mycobacterial heat shock protein 65 (hsp65). Surprisingly,
days after immunization with heat-killed Mtb, residual mycobacterial
DNA was detected in the spleen and bone marrow, but not the synovium,
of arthritic rats.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male inbred Lewis rats (RT1.B1) of 6–9 wk of age and female BALB/c mice of 6–8 wk of age were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and The Jackson Laboratory (Bar Harbor, ME), respectively. All animals were maintained in the University of California at San Diego Animal Facility (La Jolla, CA), which is accredited by the American Association for the Accreditation of Laboratory Animal Care. All experiments were approved by the Animal Subjects Committee in accordance with U.S. Department of Agriculture guidelines.
Reagents
Heat-killed Mtb (strain H37Ra) and IFA were obtained from Difco
(Detroit, MI). Hsp65 and 
-galactosidase (-gal) were obtained from
Stressgen Biotechnologies (Victoria, British Columbia, Canada) and
Sigma-Aldrich (St. Louis, MO), respectively. Phosphorothioate
ISS-containing oligodeoxynucleotide (ISS-ODN) and control ODN were
purchased from Trilink Biotechnologies (San Diego, CA). The sequence of
the ISS-ODN was 5'-TGACTGTGAACGTTCGAGATG-3', and the sequence of the
control ODN was 5'-TGACTGTGAATGTTAGAGATGA-3'. Endotoxin levels of the
ISS-ODN and control ODN were below the limit of detection (<1 ng/mg
DNA) as measured by the Limulus amebocyte lysate assay
(BioWhittaker, Walkersville, MD).
Induction and clinical assessment of experimental arthritis
AA was induced by a single intradermal (i.d.) injection at the base of the tail with 0.3 mg of Mtb suspended in 100 µl of IFA (CFA). Rats were examined daily for clinical signs of arthritis in a blinded fashion. Severity of arthritis was assessed by weight loss and scoring each paw from zero to four based on degree of swelling, erythema, and deformation of the joints (maximum score, 16).
DNase treatment of Mtb
Mtb was incubated overnight at 37°C with 5 µg/ml DNase I (Sigma-Aldrich) in the presence of 5 mM MgCl2. After extraction, DNA depletion was confirmed by agarose gel electrophoresis and ethidium bromide staining. Mtb (0.3 mg) was suspended in 100 µl of IFA (DNase/CFA).
Immunization protocols
Rats received a single i.d. injection with either Mtb, DNase-treated Mtb, or DNase-treated Mtb supplemented with either ISS-ODN (3, 10, 30, and 100 µg) or control-ODN (100 µg) in a final volume of 100 µl of IFA. A total of 3 µg of ISS corresponds to the estimated concentration of DNA in 0.3 mg of heat-killed Mtb. The animals were examined daily for clinical signs of arthritis, as described above. Animals injected with ISS-ODN (100 µg) suspended in 100 µl of IFA served as a control.
Mice received a single i.d. injection of -gal (10 µg) emulsified
with either Mtb (50 µg), DNase-treated Mtb (50 µg), or
DNase-treated Mtb supplemented with either ISS-ODN (10 µg) or control
ODN (10 µg) in a final volume of 50 µl of IFA. Injections of
-gal alone or -gal with either IFA or IFA/ISS-ODN were used as
controls. Four weeks after injection, mice were bled and spleens were
harvested for cytokine assays.
Ab and cytokine assays
Mouse -gal-specific IgG2a and IgG1 Abs were measured by ELISA
and are expressed in relative units, as previously described
(3). Ag-specific mouse splenocyte cytokine profiles also
were assessed by ELISA, as described earlier (3). Mouse
spleens were harvested 4 wk after injection, teased to single cell
suspensions, and resuspended in RPMI 1640 supplemented with 10%
heat-inactivated FCS. Splenocytes were incubated at 5 x
105 cells/well in 96-well plates in a final
volume of 200 µl of complete medium with -gal added at 10 µg/ml
at 37°C and 5% CO2. Culture supernatants were
harvested at 72 h and analyzed for IL-5 and IFN- (BD
PharMingen, San Diego, CA) by ELISA, according to the manufacturer’s
directions.
Rat inguinal lymph node cells (ILN) were harvested for Ag-specific
IFN- and RANKL assessment 55 days after induction of AA. Briefly,
ILN were strained through a 70-µm nylon cell strainer (BD
Biosciences, Mountain View, CA) and resuspended at 1 x
106 cells/well in 96-well plates in a final
volume of 200 µl of IMDM supplemented with 10% heat-inactivated FCS.
ILN were cultured for 72 h with hsp65 (10 µg/ml) and analyzed by
ELISA, as per kit instructions. IFN- was obtained from Biosource
International, Camarillo, CA; RANKL was obtained from R&D Systems,
Minneapolis, MN. The Ab against mouse RANKL cross-reacted with the rat
protein.
Detection of Mtb DNA in rat tissues
Mtb in various rat tissues was detected by PCR. Two reactions for each organ for each time point were performed for each animal (n = 4). Lewis rats were sacrificed 1, 3, 7, 10, 14, 17, 21, 29, and 36 days after receiving a single i.d. injection of 0.3 mg of Mtb in IFA. For each time point, approximately 50 mm3 of tail base (site of injection), spleen, inguinal lymph node, bone marrow of the tibia, synovium, kidney, and liver was harvested and frozen at -80°C until analyzed. Samples were boiled for 10 min in 0.5 ml of pyrogen-free sterile water to lyse cells and release DNA.
The seminested PCR assay consisted of two rounds with each reaction (total volume, 30 µl) performed as per AdvanTaq Plus PCR kit instructions (Clontech Laboratories, Palo Alto, CA). The template for the first PCR round consisted of 1 µl of a 1/20 dilution of DNA extract with a 0.3 µM concentration of forward primer (IDT, San Diego, CA) (5'-GATCCTGCGAGCGTAGGCGTCGGTGAC-3') and reverse primer (5'-GATCTCGTCCAGCGCCGCTTCGGACCA-3') (6). This primer pair was designed to amplify a 123-bp region of a Mtb multicopy insertion sequence (IS6110). The amplification parameters included 20 cycles each of denaturation at 94°C for 30 s, followed by a single annealing and extension step at 72°C for 30 s.
A second round of PCR amplification used the forward primer and an internal primer (5'-GCACCTAACCGGCTGTGGGTAGCA-3') with identical conditions and one-thirtieth of the amount of the first PCR product as a template. The second round of amplification consisted of 27 cycles and yielded a 100-bp product. All samples were compared against the control gene, actin, with an amplification procedure of 35 cycles with identical conditions for each cycle. The PCR products were analyzed by electrophoresis on 1.5% metaphor agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.
Dot-blot hybridization
Nucleic acids were extracted from the injected areas (tail base) and from affected synovial tissues (ankle) by boiling in 1 ml of Tris-EDTA buffer at 100°C for 10 min. Solid materials were removed by centrifugation at 14,000 x g for 10 min, and 1 µl from each supernatant was loaded on a Hybond N+ membrane (Amersham, Arlington Heights, IL). After fixation of the nucleic acids onto the membrane with 0.2 M NaOH, the filter was hybridized with 32P-labeled oligonucleotide complementary to the ISS-ODN (5'-TGACTGTGAACGTTCGA), washed with 2x SSC containing 0.1% SDS three times, and exposed to x-ray film (Kodak X-OMAT Blue XB-1; Kodak, Rochester, NY).
Histology
Excised rat joints were stained with H&E. A synovial histology score was determined on the stained sections using a semiquantitative scale that measures synovial inflammation (0–4), bone and cartilage erosions (0–4), marrow infiltration (0–4), and extra-articular inflammation (0–4) (maximum score, 16).
Statistics
Two-tailed unpaired Student t tests were used to compare Ab levels, cytokine levels, clinical arthritis scores, and histology scores using StatView (SAS Institute, Cary, NC) and Mathsoft computer software (Mathsoft, Cambridge, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MTb extract was treated with DNase at 37°C overnight. The digest product, untreated Mtb DNA, and a 10-bp ladder standard were electrophoresed on a 20% polyacrylamide gel and stained with cyber green. The DNase treatment was complete, as no digest products were observed (<10 bp, data not shown).
DNase treatment inhibits the Th1-skewing properties of CFA
CFA has been reported to polarize immune responses to
coadministered Ags toward a Th1 type. Before turning to the model of
AA, we questioned whether it is possible to abolish the Th1-skewing
capacities of CFA by DNase treatment and subsequently restore it with
oligonucleotides containing ISS sequences. Heat-killed Mtb was treated
with DNase and subsequently suspended in IFA to make DNase-CFA. To test
immunogenicity of DNase-CFA in vivo, we immunized BALB/c mice with
-gal and the following adjuvants: IFA, CFA, DNase-CFA, DNase-CFA
mixed with ISS-ODN, DNase-CFA mixed with control ODN, and IFA mixed
with ISS-ODN. The immunization consisted of a single i.d. injection of
-gal emulsified with one of the mentioned adjuvants. Four weeks
later, blood and spleens were harvested. The Ab isotype responses to
-gal were assessed (Fig. 1
, upper panel). Compared with the Ag-CFA combination, mice
immunized with -gal and DNase-treated CFA had reduced IgG2a levels
(p = 0.05). However, when DNase-treated CFA was
mixed with ISS-ODN, anti--gal IgG2a production was restored
(p < 0.0001).
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and IL-5
by splenocytes after immunization with -gal and the different
adjuvants. The results mimicked the Ab data (Fig. 1
, lower
panel). Mice immunized with -gal and DNase-treated CFA showed
reduced -gal-specific IFN- levels, compared with the CFA-positive
control (p = 0.08). In contrast, after
immunization with -gal and DNase-treated CFA and ISS-ODN, a strong
increase in IFN- production was found, in comparison with -gal
DNase-treated CFA alone or with control ODN (p
< 0.02), indicative of a Th1-like response. A similar response was
induced when -gal was coadministered with IFA mixed with ISS-ODN,
whereas -gal coadministered with DNase-CFA and control ODN did not
lead to increased production of IFN- (p <
0.03). Thus, DNase treatment of Mtb reduced its Th1-polarizing adjuvant
effect, which could be completely restored with ISS-ODN alone. ISS determine the severity of AA
Next, we determined whether the presence of Mtb DNA is necessary
to induce AA. We first treated heat-killed Mtb with DNase until no
high-m.w. DNA was detectable by electrophoresis and ethidium bromide
staining. We injected Lewis rats with either emulsified heat-killed
Mtb, DNase-treated Mtb, DNase-treated Mtb supplemented with ISS-ODN (3,
10, 30, and 100 µg), or DNase-treated Mtb with a control ODN. The
results are shown in Fig. 2. DNase
treatment of Mtb led to a delay in the onset and a marked reduction in
the severity of arthritis (mean maximum arthritis score, 5) compared
with the positive control (mean maximum arthritis score, 12;
p < 0.0001). The addition of ISS-ODN (100 µg) to
DNase-treated Mtb restored the severity of arthritis completely (mean
maximum arthritis score, 12), whereas the addition of control ODN was
devoid of efficacy (p < 0.0001). The clinical
findings were also reflected in the histological scores (Fig. 3). These experiments showed that the
severity of joint inflammation in AA depends on the presence of
Mtb DNA.
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and 3). These results suggest that mycobacterial Ags, or other factors
besides DNA, also are required for arthritis induction. Immunostimulatory DNA induces Th1 responses and the production of soluble RANKL to Mtb Ags
To determine the effects of Mtb DNA on Ag-specific cytokine
synthesis, we cultured ILN at day 55 after arthritis induction with
medium alone, Mtb, DNase-treated Mtb, or purified Mtb hsp65. After a
72-h culture, supernatants were collected, and IFN- and RANKL levels
were assayed. Fig. 4 illustrates the
results of hsp65 stimulation. Similar results were found with Mtb
extracts (data not shown). Lymph node cells from rats immunized with
whole heat-killed Mtb produced IFN- and soluble RANKL after in vitro
activation with hsp65. In contrast, lymph node cells from rats
immunized with DNase-treated Mtb produced significantly less IFN-
and RANKL after in vitro restimulation with hsp65
(p < 0.05). However, lymph node cells from
rats immunized with DNase-treated Mtb, which had been supplemented with
ISS-ODN, produced high levels of IFN- and RANKL after in vitro
activation with hsp65, whereas lymph node cells from rats immunized
with DNase-treated Mtb, mixed with a control ODN, produced IFN- and
RANKL at levels comparable with those produced by cells from rats
immunized with DNase-treated Mtb (p < 0.05,
DNase-CFA/ISS-ODN vs DNase-CFA/control ODN). We could not detect
Ag-specific IFN- and RANKL production in naive rats. Other
experiments demonstrated no significant production of IL-4 or IL-10 in
any of the lymph node cultures. Thus, primed lymph node cells from rats
immunized with a mixture containing ISS (either Mtb DNA or ISS-ODN)
produced significantly higher levels of IFN- and RANKL, compared
with primed lymph node cells from rats immunized with Mtb without ISS.
The levels of IFN- and RANKL production by Ag-restimulated lymph
node cells correlated with the severity of arthritis found in the
different treatment groups.
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Because intra-articular injections of ISS induce joint
inflammation, it was important to determine whether Mtb DNA was present
in the joints. At 1, 3, 7, 10, 14, 17, 21, 29, and 36 days after
injection of Mtb into the tails of Lewis rats, PCR analyses were
performed on tissue samples taken from kidney, liver, spleen, bone
marrow, base of the tail, inguinal lymph node, and synovium (Fig. 5). Mycobacterial DNA was detected at the
site of injection, in the spleen up until day 36, in the draining
(inguinal) lymph nodes at day 10, and in the bone marrow at days 3, 7,
10, and 14. No Mtb DNA was detected in the liver and kidney, or
synovium. Thus, after immunization with CFA, mycobacterial DNA
disperses to bone marrow and lymphoid tissues, but not to the synovium.
Hence, the arthritogenic effects of Mtb cannot be attributed to a local
effect on the synovium, as was the case in the model of septic
arthritis (5).
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B, ISS-ODN was detected in the area of
injection but not in the affected synovium. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Once established, AA is clearly a T cell-mediated process, because the
passive transfer of CD4+ T cells from affected
rats to irradiated syngenic recipients induces disease, whereas serum
transfer has no deleterious effects (10). Moreover,
arthritis can be triggered by a cross-reactive CD4 T cell clone that
recognizes an epitope present on bacterial heat shock proteins and
cartilage proteoglycans (11, 12). However, the early
induction of AA requires stimulation of innate immunity, as indicated
by the ability of cyclooxygenase inhibitors, TNF- antagonists, and
chemokine blockers to prevent disease (13, 14, 15).
One way to explain these data is to propose that the activation of innate immunity by ISS primes the joints for the subsequent development of a Th1 autoimmune response. In this context, Sato et al. (16) recently reported that the prior injection of ISS-ODN into mice exacerbated the severity of arthritis induced by immunization with collagen in CFA. Unlike most other bacterial products that activate innate immunity, ISS are poorly immunogenic and can persist for long periods within transfected cells. Tarkowski et al. (5) found that the direct injection of bacterial DNA into the articular cavity of rats induced severe inflammation. Both Ag-Ab complexes and systemically administered Ags have been shown to traffic to the joints. Thus, the systemic dispersal of Mtb DNA, and its subsequent trapping in the synovium, could explain the potentiating effects of ISS in AA. However, from 1–36 days after injection of heat-killed Mtb in the tail, we were unable to detect Mtb DNA sequences or ISS-ODNs within the joints. Instead, the DNA dispersed primarily to the bone marrow, the lymph nodes, and the spleen.
The activation of innate immune responses within the bone marrow may be particularly relevant to the induction of arthritis. In the collagen arthritis model, which requires Freund’s adjuvant, the normally small channels between the bone marrow and synovial cavity enlarge before the onset of joint swelling (17). The resorption of subchondral bone is an established early event in AA. Bone resorption requires the activation of osteoclast precursors within the marrow. The osteoclast activation, in turn, depends upon the increased production of RANKL (osteoprotegerin ligand/TNF-related apoptosis-inducing ligand) by T lymphocytes. Kong et al. (18) discovered that inhibition of RANKL signaling prevented bone and cartilage destruction in the AA model, although leukocyte infiltration into the joints was still present. Our experiments showed that increased RANKL production by T cells is part and parcel of the immune system activation induced by ISS and Ag.
The deposition of Mtb DNA in the bone marrow not only argues against the direct involvement of Mtb DNA in the pathogenesis of AA, but also suggests that other Mtb-related compounds (e.g., hsp65) could follow the same course. Activation of T cells in the bone marrow by Mtb-related Ags might induce the local secretion of RANKL, which activates osteoclasts and thus makes the joint permeable and more susceptible to the influx of inflammatory cells and the subsequent development of arthritis.
Recently, there has been a revival of interest in the role of innate immunity in the pathogenesis of human rheumatoid arthritis and other inflammatory forms of arthritis of unknown etiology. The products of activated macrophages are abundant in inflammatory synovial fluids. Both bacterial DNA and peptidoglycans have been detected in the joints (19, 20). Th1-type responses to bacterial heat shock proteins are readily measurable in synovial fluid T lymphocytes, whereas responses to other Ags are often weak or absent (21, 22). Moreover, endogenous hsp65, as expressed in inflamed synovial tissue (23), can trigger innate immune responses through the LPS receptor Toll-like receptor (TLR)4 and CD14 (24).
The nine known TLRs regulate the response of the innate immune system to bacterial products (25). Knockout experiments have demonstrated that the murine response to ISS-ODN requires TLR9 (26). In addition to stimulation through TLR9, various Mtb products such as lipopeptide or hsp65 also can interact with TLR2 and TLR4, respectively (25, 27). Thus, the activation of innate immunity by Mtb is multifactorial. Consistent with this interpretation, DNA digestion of mycobacteria did not totally abolish its arthritis-promoting properties. Future experiments will need to determine the role of different TLRs in the human response to microbial DNA, the expression of TLRs in cells from the bone marrow and joints, the fate of bone marrow-derived macrophages exposed to bacterial products, and the interactions between the TLRs and various cytokines.
In general, phosphorothioate ODN are much more potent than phosphodiester ODN or intact DNA. Thus, the lower efficacy of ISS-ODN to induce AA could suggest that intact Mtb DNA is much more effective than the comparable amount of ISS-ODN. This could reflect some intrinsic property of Mtb. Alternatively, the DNase treatment may have destroyed some other factor that plays a proinflammatory role in this system.
Bacterial DNA and ISS-ODN are much more potent activators of the innate and adaptive immune system in rodents, compared with primates (1). However, various ILs have been demonstrated to synergize with ISS in the activation of human NK cells (28). The confined synovial spaces of patients with inflammatory arthritis can act as a sink for cytokines and other inflammatory mediators. In this setting, bone marrow-derived macrophages, lymphocytes, and NK cells could be more readily stimulated by bacterial DNA. Thus, although ISS are unlikely to be a direct cause of rheumatoid arthritis, they could play a significant role in disease progression and severity. Therefore, inhibitors of ISS binding to TLR9 or of ISS-induced signal transduction might be useful for the treatment of affected patients.
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Acknowledgments |
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Footnotes |
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2 A.R. and B.J.P. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Dennis A. Carson, Department of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0663. E-mail address: dcarson{at}ucsd.edu
4 Abbreviations used in this paper: ISS, immunostimulatory DNA sequence; AA, adjuvant arthritis; -gal, -galactosidase; hsp65, mycobacterial heat shock protein 65; i.d., intradermal; ILN, inguinal lymph node cell; Mtb, Mycobacterium tuberculosis; ODN, oligodeoxynucleotide; RANKL, receptor activator of NF-B ligand; TLR, Toll-like receptor.
Received for publication May 23, 2001. Accepted for publication October 29, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synergistically inhibit adjuvant arthritis in Lewis rats. Cell. Mol. Life Sci. 57:1457.[Medline]
production in vitro. J. Immunol. 163:3642.This article has been cited by other articles:
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