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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

To explore the effect of bortezomib in splenocytes and fibroblast-like synoviocytes (FLS) and its in vivo potency in a rat model of adjuvant-induced arthritis (AIA), which resembles human rheumatoid arthritis (RA).

Methods

AIA was induced with Freund's complete adjuvant. Splenocyte and FLS proliferation and apoptosis were measured by radioactivity incorporation and flow cytometry, respectively. The invasiveness of FLS from rats with AIA was tested in a Transwell system. The pattern of cytokine secretion was evaluated by cytometric bead array in splenocyte supernatants. Bortezomib was administered prophylactically or therapeutically, and arthritis was assessed clinically and histologically. Immunohistochemistry was performed for markers of inflammation and angiogenesis in joints. Hematologic and biochemical parameters were tested in peripheral blood (PB). Representative animals were examined by computed tomography (CT) scanning before and after bortezomib administration. The expression of Toll-like receptor 2 (TLR-2), TLR-3, and TLR-4 in PB and FLS was measured by real-time polymerase chain reaction, and alterations in specific cell populations in PB and spleen were determined by flow cytometry.

Results

In vitro, bortezomib exhibited significant inhibitory and proapoptotic activity in splenocytes and FLS from rats with AIA, altered the inflammatory cytokine pattern, and reduced the invasiveness of FLS from rats with AIA. In vivo, bortezomib significantly ameliorated disease severity. Remission was associated with improved histology and decreased expression of CD3, CD79a, CD11b, cyclooxygenase 1, and factor VIII in target tissues as well as down-regulation of TLR expression in PB and cultured FLS. CT scanning demonstrated a bone healing effect after treatment.

Conclusion

Our findings suggest that bortezomib affects AIA in a pleiotropic manner and that this drug may be effective in RA.

Bortezomib is the first proteasome inhibitor that has been introduced in the clinic for the treatment of multiple myeloma (1–3). By inhibiting NF-κB function, bortezomib treatment results in increased tumor cell sensitivity to chemotherapy (4, 5) and induction of apoptosis of leukemic (6) and alloreactive (7) T cells. Because NF-κB activation results in the transcription of proinflammatory molecules (8), it has been speculated that NF-κB inhibition may also ameliorate inflammatory and autoimmune conditions (9, 10).

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammation and joint destruction (11). The pain, swelling, stiffness, and tissue destruction that accompany inflammatory disease result from a cascade of events that is initiated and propagated by the production of cytokines and chemokines and the cell surface expression of cell adhesion molecules (11). RA represents a potential disease target for proteasome inhibitors because the most important proinflammatory mediators (tumor necrosis factor α [TNFα], interleukin-1 [IL-1], IL-6, and vascular cell adhesion molecule 1) are regulated by NF-κB (9, 12, 13). NF-κB1, the predominant subunit from the Rel/NF-κB family, is expressed in the cartilage–pannus junction (14), which is the site of joint erosion in RA.

NF-κB1–deficient mice are resistant to the induction of acute and chronic inflammatory arthritis (15). NF-κB activation prior to the onset of clinical manifestations of arthritis has been shown in both murine type II collagen–induced arthritis and rat adjuvant-induced arthritis (AIA) (16, 17), two models of inflammatory arthritis.

In the present study, we demonstrated that NF-κB inhibition by bortezomib significantly ameliorates AIA through antiproliferative and proapoptotic effects in splenocytes and fibroblast-like synoviocytes (FLS), finally resulting in reduced inflammation and bone healing.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Animals.

Seven-week-old Fisher F344/N rats weighing 140–200 gm were used. All rats were housed under standard laboratory conditions (at 22°C under 12-hour light/12-hour dark conditions). The study was approved by the Animal Care and Use Committee of the regional veterinary health authority.

Reagents.

Bortezomib (PS-341; Velcade) was kindly provided by Janssen-Cilag. Aliquots of 1 mg/ml were prepared in Dulbecco's phosphate buffered saline (PBS) and stored at −80°C until used.

Induction of AIA.

Arthritis was induced in rats by intradermal injection, into the base of the tail, of 0.1 ml of Freund's complete adjuvant (CFA) (containing 0.6 mg of heat-inactivated Mycobacterium butyricum [Difco 264010]). The first signs of inflammation started to develop 11–13 days later.

Experimental design.

Bortezomib was administered before the onset of symptoms (days 3, 6, 9, and 12), early after the onset of symptoms (days 13, 16, 19, and 22), and in established disease (days 18, 21, 24, and 27), at a dose of 0.25 mg/kg intraperitoneally twice a week for 2 weeks. Control animals received intraperitoneal injections of an equal volume of PBS at the same time points. Animals were killed 3–7 days after the last injection, and tissue specimens (from blood, spleens, joints, and synovium) were collected for further studies. All experiments were performed at least 3 times with 5 rats per group.

Assessment of arthritis.

The total arthritis index was assessed on a 4-point scale per limb, with a maximum score of 16 for each rat, where 0 = no erythema or swelling, 1 = slight erythema or swelling of 1 joint, 2 = swelling of ≥2 joints, 3 = erythema or swelling of all joints, and 4 = ankylosis and deformity of the joint. Grading was performed daily by 2 independent observers (A. Papadopoulou and A. Paraskeva).

Histopathologic analysis and immunohistochemistry.

Spleens and paws were removed for histopathologic examination. Paraffin-embedded sections (4–6 μm thick) were cut along a longitudinal axis at varying depths for each ankle after routine fixation. Histopathologic changes were evaluated in a blinded manner by a pathologist (EA) and scored on a scale of 1–4 per parameter, with a maximum score of 20 for each affected joint. The following 5 parameters were evaluated: inflammatory cell infiltration, synovial hyperplasia, pannus formation, cartilage damage, and bone resorption. For inflammatory cell infiltration, 1 = normal appearance, 2 = mild sublining synovial fibrosis and cell infiltration, 3 = moderate sublining synovial fibrosis, edema, and cell infiltration, and 4 = marked periarticular inflammatory infiltration and synovial fibrosis. For synovial hyperplasia, 1 = 2 layers (normal appearance), 2 = 3 layers, 3 = 4 layers, and 4 = >4 layers. For pannus formation, 1 = normal appearance, 2 = presence of vascularized connective tissue at the edges of the joint, 3 = vascularized connective and granulomatous tissue confined to the joint, and 4 = vascularized connective and granulomatous tissue penetrating muscles and soft tissue. For cartilage damage, 1 = normal appearance, 2 = mild articular cartilage damage, 3 = moderate cartilage damage with abnormal formations consisting of dead chondrocytes (abnormal osteochondroplasia), and 4 = joint ankylosis, complete loss of cartilage, and marked osteochondroplasia. For bone resorption, 1 = normal appearance, 2 = bone resorption at the bone margins, 3 = bone resorption involving the subchondral bone but sparing the cartilage and bone interface, and 4 = marked bone resorption involving the cartilage and bone interface.

Joint sections were sequentially incubated with antibodies against CD3 (at a dilution of 1:60; Serotec), CD79a (at a dilution of 1:100; Acris Antibodies), CD11b (at a dilution of 1:30; Acris Antibodies), factor VIII (FVIII) (at a dilution of 1:800; Abcam), and cyclooxygenase 1 (COX-1) (at a dilution of 1:70; Santa Cruz Biotechnology), followed by secondary biotinylated antibody or polymer (Envision Dako) with horseradish peroxidase enzyme substrate. Pretreatment was performed according to the recommendations of the manufacturer. All tissue samples were counterstained with hematoxylin.

FLS culture.

Synovial cells were isolated under sterile conditions. Synovial tissue was digested with type VIII collagenase (1 mg/ml; Sigma) for at least 2 hours at 37°C and then filtered through a 100-μm filter. Cells were subsequently incubated in a humidified 5% CO2 atmosphere at 37°C in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mML-glutamine. Cells grown to confluence were detached with trypsin containing 0.25% EDTA (Gibco) and split at a 1:3 ratio. The cells were identified morphologically by their homogeneous bipolar appearance and by immunocytochemistry for prolyl 4-hydroxylase (Acris Antibodies) expression in cytospin preparations (data not shown). Synoviocytes from passages 3–5 were used for the in vitro experiments.

Splenocyte and FLS proliferation assay.

Rat spleens were removed aseptically 9–15 days after CFA injection, and single-cell suspensions were prepared by passage through a 70-μm filter. The cells were washed twice with PBS and cultured in the presence of increasing concentrations of bortezomib. Splenocytes were plated in triplicate at 2 × 105 cells/well in 96-well plates in a total volume of 0.2 ml RPMI medium containing 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mML-glutamine, and 50 μM 2-mercaptoethanol, with or without 0.5 μg/ml of concanavalin A (Con A). The plates were incubated at 37°C in a humidified atmosphere of 5% CO2 for 3 days. Cells were pulsed with 0.4 μCi 3H-thymidine/well 18 hours before the end of culture and harvested with a cell harvester (Skatron MCH 1).

FLS were seeded at a density of 20,000 cells/well (in a 24-well plate) and cultured in 1 ml FLS medium (as described above) in which bortezomib was added for increasing culture time. After 3 days, 0.5 μCi 3H-thymidine/well was added, and plates were incubated at 37°C for 4 hours. After sequential washes with 1 ml PBS, 1 ml 10% trichloroacetic acid (Applichem), and 1 ml PBS, 0.1 ml of 0.3M NaOH was added to each well, and cells were incubated for 30 minutes at room temperature.

The incorporated radioactivity in splenocytes and FLS was measured with a scintillation counter (Wallac 1409). The proliferative response is presented as absolute counts per minute. All experiments were performed independently at least 3 times for each condition described.

Invasion assay.

The invasiveness of FLS from rats with AIA was evaluated in a Transwell system (6.5 mm diameter, 8.0 mm; Costar). The Matrigel (Matrigel basement membrane matrix; Becton Dickinson) was diluted to 0.375 mg/ml in IMDM. The Transwells were preincubated with 300 μl of IMDM for 30 minutes at 37°C and, after removal of the medium, coated overnight with 300 μl of 0.375 mg/ml Matrigel in IMDM in a functioning laminar flow cabinet. The next day, coated wells were preincubated with 300 μl of IMDM for 1 hour at 37°C, and then 2 × 104 FLS in fresh IMDM were seeded in the inner well. IMDM/20% FBS with or without 10 nM bortezomib was added in the outer wells. After 3 days of incubation, the cells were fixed with 4% paraformaldehyde in PBS for 20 minutes, washed with PBS, and stained with hematoxylin and eosin. The cells that crossed the matrix and the Transwell membrane were observed under a light microscope.

Fluorescence-activated cell sorting (FACS) analysis.

Peripheral blood (PB) and spleen cells, after erythrocyte lysing, were stained using fluorescein isothiocyanate–conjugated or phycoerythrin (PE)–conjugated mouse monoclonal antibodies against CD4, CD8, and CD11b (Becton Dickinson). Nonspecific binding was corrected with isotype-matched controls. The absolute numbers of CD4+, CD8+, and CD11b+ cells were calculated based on the percentages of these cells, determined by flow cytometry, and the individual cell counts in blood and spleen.

FLS and Con A–stimulated splenocytes were cultured in the presence or absence of bortezomib for 3 days. Apoptotic cells were measured by FACS analysis as annexin V–positive/7-aminoactinomycin D (7-AAD)–negative events, using an annexin V apoptosis detection kit (Becton Dickinson). Results were obtained on a FACSCanto flow cytometer (Becton Dickinson) and analyzed with the FACSDiva 6 software. The pattern of cytokine secretion in supernatants from Con A–stimulated splenocytes, cultured in the presence or absence of bortezomib for 3 days, was evaluated by FACS for the detection of soluble analytes (interferon-γ [IFNγ], TNFα, and IL-6) (BD Cytometric Bead Array).

Real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR).

Total RNA was isolated using the QIAmp RNA blood mini kit (Qiagen), reverse-transcribed with the RT2 First Strand kit (Superarray Biosciences), and amplified by real-time PCR using the ABI 7500 detection system (Applied Biosystems) and RT2 quantitative PCR primers and RT2 quantitative PCR Master Mix (Superarray Biosciences). The endogenous control GAPDH was used for correcting the results with the Ct method for relative quantification. The differences in the Ct values of the sample and GAPDH were calculated (ΔCt). Relative expression levels were calculated using the formula ΔΔCt = ΔCt(treated) − ΔCt(control), and the value used to plot the relative expression was calculated using the expression 2−ΔΔCt.

Complete blood cell counts and biochemical testing.

When rats were killed, PB from control and bortezomib-treated animals was examined for white blood cell count, hemoglobin, hematocrit, platelet count, and levels of aspartate aminotransferase, alanine aminotransferase, bilirubin, blood urea nitrogen, and creatinine to assess any potential in vivo toxicity of bortezomib at the administered dose.

Computed tomography (CT) scanning.

The knee and ankle joints from 2 representative rats were examined by CT (Multislice 4 Sensation; Siemens). Images of 1-mm slice thickness were obtained and then reconstructed at 1-mm slice thickness.

Statistical analysis.

Results were expressed as the mean ± SEM. Changes within treatment groups were analyzed by one-way analysis of variance (ANOVA). Changes between treatment groups were analyzed by two-way ANOVA (interaction treatment and dose). Analyses were followed by Tukey's post hoc test for multiple comparisons. Statistical comparisons between 2 groups were analyzed using Student's t-test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Inhibition of the proliferation of splenocytes and increase in T cell apoptosis in vitro by bortezomib treatment.

To investigate whether bortezomib affects splenocyte proliferation, Con A–activated or resting splenocytes from normal rats or rats with AIA were cultured in the presence of increasing concentrations of bortezomib for 72 hours or of 10 nM bortezomib for increasing culture time. Bortezomib at 5 nM significantly inhibited the proliferation of resting splenocytes from rats with AIA, whereas 10 nM bortezomib was needed to inhibit the proliferation of resting normal splenocytes (P < 0.001), suggesting that activated cells are more susceptible to inhibition by bortezomib (Figure 1A, left panel). Bortezomib at a dose equal to or higher than 5 nM significantly suppressed the proliferation of Con A–activated splenocytes from both normal rats and rats with AIA (P < 0.001) (Figure 1A, right panel). Significant inhibition of normal splenocytes and splenocytes from rats with AIA, either resting or Con A–activated, was detected from the first 24 hours of culture with 10 nM bortezomib (P < 0.001) (Figure 1B). Consistent with the results of previous studies showing that RA T cells ex vivo are paradoxically hyporesponsive to stimulation by mitogens (18, 19), proliferation of splenocytes from rats with AIA was significantly reduced compared with normal splenocyte proliferation (∼2-fold reduction) (P = 0.03) (Figures 1A and B).

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Figure 1. Splenocyte and fibroblast-like synoviocyte (FLS) proliferation and apoptosis in cells from normal rats and rats with adjuvant-induced arthritis (AIA). A, Significant inhibition of resting splenocytes (left) and concanavalin A (Con A)–activated splenocytes (right) from normal rats or rats with AIA by increasing concentrations of bortezomib in a 72-hour culture. B, Significant inhibition of resting splenocytes (left) and Con A–activated splenocytes (right) from normal rats or rats with AIA after addition of 10 nM bortezomib to the culture for 24, 48, or 72 hours. C, Significant increase in the apoptosis of normal T cells and T cells from rats with AIA at bortezomib concentrations equal to or higher than 100 nM. D, Significant inhibition of FLS proliferation after addition of 10 nM bortezomib for 24, 48, or 72 hours in cultured normal FLS or FLS from rats with AIA. E, Significant increase in the apoptosis of normal FLS and FLS from rats with AIA at bortezomib concentrations equal to or higher than 50 nM. F, Invasion capacity of FLS in a Transwell system. The comparable invasiveness of normal FLS (I) and FLS from rats with AIA treated with 10 nM bortezomib (III) in contrast to increased numbers of invading FLS from rats with AIA (II) is shown. Proliferation is presented as absolute cpm. Bars in AE show the mean and SEM. ∗ = P < 0.05 versus normal untreated cells; ∗∗ = P ≤ 0.001 versus normal untreated cells; # = P < 0.05 versus untreated cells from rats with AIA; ## = P ≤ 0.001 versus untreated cells from rats with AIA, by Tukey's test; $ = P < 0.008. 7-AAD = 7-aminoactinomycin D.

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To investigate whether bortezomib affects the apoptosis of Con A–activated T lymphocytes from normal and arthritic rats, we used annexin V and 7-AAD staining by flow cytometry. Apoptotic T cells were determined as annexin V–positive and 7-AAD–negative events in the CD3+ cell population. As shown in Figure 1C, the percentage of CD3+/annexin V–positive/7-AAD–negative cells from normal or arthritic animals significantly increased at drug concentrations equal to or higher than 100 nM (P = 0.006 for normal animals and P = 0.01 for rats with AIA) (Figure 1C).

Inhibition of the proliferation of FLS, increase in the apoptosis of FLS, and attenuation of FLS invasiveness in vitro by bortezomib.

To investigate whether bortezomib affects FLS, which represent a critical cell population for the initiation and propagation of arthritis, we tested its effect on the proliferation, apoptosis, and invasion of FLS. Bortezomib significantly inhibited the proliferation of normal FLS and FLS from rats with AIA when added at 10 nM for 24, 48, or 72 hours in culture (P < 0.001) (Figure 1D). Not unexpectedly, in the absence of bortezomib (at 0 hours) (Figure 1D), FLS from rats with AIA demonstrated increased proliferative activity compared with normal FLS (P = 0.0009), providing support for the concept that synovial hyperplasia in RA is at least partly mediated by increased FLS proliferation.

Bortezomib increased the apoptosis of normal FLS at concentrations higher than 100 nM, whereas concentrations of 50 nM and higher significantly increased the apoptotic activity of FLS from rats with AIA (P < 0.001), as assessed by PE-conjugated annexin V/7-AAD staining and flow cytometric analysis (Figure 1E). Interestingly, although the apoptosis of FLS from rats with AIA did not differ significantly from that of normal FLS, the proapoptotic effect of bortezomib at the highest concentrations tested (100 nM and 1,000 nM) was stronger in FLS from rats with AIA (P < 0.008) (Figure 1E), suggesting again that activated cells are more susceptible to bortezomib-induced cell death.

RA is characterized by a “tumor-like” phenotype of synoviocytes, and RA FLS, unlike normal FLS, invade and degrade cartilage (20). The invasiveness of FLS from rats with AIA was tested in an in vitro invasion assay; the addition of 10 nM bortezomib in arthritic FLS seeded on Matrigel-coated Transwell filters reduced the number of invading cells compared with control invasion (arthritic FLS not treated with bortezomib), to levels similar to normal invasion (Figure 1F). The 10 nM dose was chosen for the assay, since only doses equal to or higher than 50 nM were shown to significantly affect apoptosis of FLS from rats with AIA (Figure 1E). Consequently, the decreased invasiveness of arthritic FLS in the presence of bortezomib was not the effect of direct cytotoxicity of the drug.

Reduction in the levels of inflammatory cytokines in the supernatants of splenocyte cultures treated with bortezomib.

To evaluate whether bortezomib could alter the pattern of cytokine secretion by normal or arthritic splenocytes, we collected the supernatants of Con A–activated normal and arthritic splenocyte cultures, which were supplemented with 0, 10, or 100 nM bortezomib, and measured the levels of IFNγ, TNFα, and IL-6 using a cytometric bead array. Bortezomib doses equal to or higher than 10 nM down-regulated IFNγ expression (P < 0.001) and TNFα expression (P = 0.001), and doses of 100 nM significantly reduced the levels of IL-6 (P = 0.006), in supernatants from arthritic splenocytes (Figure 2A). Cytokine secretion by normal splenocytes was either not significantly affected (IL-6) or was reduced only at the highest doses of bortezomib (IFNγ and TNFα) (Figure 2A).

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Figure 2. A, Down-regulation of interferon-γ (IFNγ) and tumor necrosis factor α (TNFα) expression after treatment with bortezomib (Bzb) in doses equal to or higher than 10 nM, and significant reduction in levels of interleukin-6 (IL-6) supernatants in splenocytes from rats with adjuvant-induced arthritis (AIA) after treatment with 100 nM bortezomib. Bars show the mean and SEM. ∗ = P < 0.05 versus untreated normal cells; # = P < 0.05 versus untreated cells from rats with AIA; ## = P < 0.001 versus untreated cells from rats with AIA, by Tukey's test. B, A representative bortezomib-treated animal before and after treatment. Note the significant remission of the redness, soft tissue swelling, and joint deformity. The total arthritis score for this animal was 8 before treatment and 1 after treatment. C, Arthritis scores in untreated rats with AIA, rats treated with bortezomib before the onset of arthritis, rats treated with bortezomib during early AIA, and rats treated with bortezomib during established AIA. Values are the mean ± SEM score from day 11 until the day rats were killed. Significant differences were observed in the arthritis score between untreated rats with AIA and bortezomib-treated groups in all conditions tested. # = P < 0.05; ## = P = 0.0004, versus untreated rats with AIA.

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Amelioration of arthritis at all disease stages by in vivo proteasome inhibition.

We observed a rather narrow therapeutic window for bortezomib-treated animals. Bortezomib at 1 mg/kg, 0.75 mg/kg, and 0.5 mg/kg proved to be highly toxic, resulting in >50% mortality, whereas bortezomib at 0.15 mg/kg had no effect (data not shown). The 0.25 mg/kg dose, administered intraperitoneally twice a week for 2 weeks, was well tolerated with no overt signs of toxicity, and it was used in all subsequent experiments.

Bortezomib treatment was started before the onset of arthritis (on days 3, 6, 9, and 12), at the onset of arthritis (on days 13, 16, 19, and 22), and in established disease (on days 18, 21, 24, and 27). Animals were killed on days 19, 25, or 29 depending on the condition tested (before AIA, during early AIA, or during late AIA, respectively).

There was a significant delay in arthritis onset and a milder clinical picture in the group treated before the onset of arthritis compared with the control group (P < 0.02). Disease severity was reduced in the group treated with bortezomib at the onset of arthritis (P < 0.05) and dramatically improved when bortezomib was administered in advanced disease (P < 0.003) as compared with the untreated AIA group (Figure 2C). Treated animals exhibited significantly reduced arthritis scores and a recovered mobility accompanied by clinical remission of the redness, soft tissue swelling, and joint deformity (Figure 2B).

Improved histopathology in the joints of bortezomib-treated animals.

Disease severity, as determined by histopathologic examination of the joints from bortezomib-treated and control animals, was consistent with the clinical evaluation of arthritis in the corresponding animals. Massive synovial thickening, pannus formation, extensive inflammatory cell infiltration, and cartilage and bone erosion were evident in joint sections from control rats (Figure 3A, part II). In contrast, there was markedly decreased inflammatory infiltration, little or no bone erosion, and limited pannus formation in the joints of bortezomib-treated rats (Figure 3A, part III). The joints of bortezomib-treated rats had a mean total histologic score of 6, compared with 14.8 in the vehicle-treated rats (P < 0.001) (Figure 3B). Immunohistochemical analysis of the ankle joints of bortezomib-treated rats demonstrated limited T lymphocyte, B lymphocyte, and macrophage cell infiltration, decreased expression of COX-1, and reduced vessel density compared with control animals (Figures 3C and 4A–D).

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Figure 3. A, Histologic assessment of the joints. I, Typical appearance of the articular cartilage (C), subchondral bone (B), synovial cells (S), and synovial membrane (SM) in a normal joint. A 2-layered normal synovial membrane is shown (inset). II, Enormous thickening and swelling of the synovial membrane in an arthritic joint due to excessive infiltration consisting of macrophages, fibroblast-like synoviocytes, lymphocytes, and neutrophils (inset). Hyperplastic synovial intima with sublining fibrosis is shown (inset). Cartilage degradation, bone erosions, and abnormal osteochondroplasia (OC-BE) are evident. An extensive vascularized granulomatous formation is seen in cartilage–pannus junctions (CPJ). III, Almost normal joint architecture with marked reduction of inflammatory cell infiltration and without cartilage damage or bone erosion in a bortezomib-treated rat. Mild sublining fibrosis and multilayered hyperplastic synovial villi can still be seen (inset). (Original magnification × 200; × 400 in insets.) B, Histopathologic scores in normal rats, untreated rats with AIA, and rats treated with bortezomib. Bortezomib-treated animals had significantly lower scores than arthritic rats (P < 0.001 by Tukey's test). Horizontal lines show the mean; circles represent scores in individual rats. C, Cyclooxygenase 1 (COX-1) expression, determined by immunohistochemistry. In the normal synovial membrane, COX-1 expression was limited in endothelial cells and synoviocytes (I), whereas in the arthritic synovium, COX-1 was universally expressed (II). After bortezomib treatment, there was decreased COX-1 expression by the inflammatory cells with a similar distribution of expression as in the normal synovial membrane (III). (Original magnification × 200.) See Figure 2 for other definitions.

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Figure 4. Immunohistochemistry in joints. I, Normal joints. Distribution of T cells (CD3+) (A), B cells (CD79a+) (B), and macrophages (CD11b+) (C) in the synovial membrane and normal vascularization (factor VIII+ [FVIII+]) (D) are shown. II, Joints from rats with adjuvant-induced arthritis. Abundant T cell accumulation (A) was evident in subsynovial and perivascular areas invading the synovial epithelium. Chronic inflammatory cell infiltrates consisting of T lymphocytes (A), B lymphocytes (B), plasma cells, and macrophages (C) were observed even in periarticular soft tissue. Macrophages were arranged within the pannus or scattered in the synovial membrane (C). Numerous small vessels were apparent not only in the highly vascularized pannus but also in sublining synovial sites (D). III, Joints from bortezomib-treated animals. Dramatically reduced inflammatory cell infiltration (AC) was seen, with limited pannus formation and decreased vascularization similar to that in normal synovium (D). (Magnification × 100 in A, B, C, and DII; × 200 in DI and DIII.)

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Attenuation of disease severity in bortezomib-treated rats, confirmed by CT imaging.

Two bortezomib-treated rats were examined by CT scanning before and after treatment. Before drug administration, CT imaging demonstrated marked soft tissue swelling and focal osteopenia or bone erosions, which reversed after treatment with bortezomib. A representative animal is shown in Figure 5A.

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Figure 5. A, Computed tomography imaging of the knee and ankle joints of a representative rat before and after bortezomib treatment. Complete remission of the soft tissue edema (blue arrow) and healing of bone erosions (red arrows) are shown. B and C, Alterations in the numbers of CD4+, CD8+, and CD11b+ cells, assessed by flow cytometry, in the peripheral blood (PB) (B) and spleen (C) of normal rats, untreated rats with AIA, and bortezomib-treated rats. Bars show the mean and SEM. ∗∗ = P ≤ 0.001 versus normal rats; ## = P ≤ 0.001 versus untreated rats with AIA, by Tukey's test. D, Toll-like receptor-2 (TLR-2), TLR-3, and TLR-4 expression, determined by real-time reverse transcriptase–polymerase chain reaction, in PB and fibroblast-like synoviocytes (FLS) from normal rats, untreated rats with AIA, and bortezomib-treated rats. Rats with AIA were killed on day 16 or 31. Bortezomib-treated rats were killed on day 31. Expression of TLR-2, TLR-3, and TLR-4 in PB and cultured FLS from bortezomib-treated rats was decreased compared with that in rats with AIA killed on day 31. Bars show the mean and SEM. ∗ = P < 0.05 versus normal rats; # = P < 0.05 versus rats with AIA assessed on day 31; ∗∗ = P ≤ 0.001 versus normal rats; ## = P ≤ 0.001 versus rats with AIA assessed on day 31, by Tukey's test. See Figure 2 for other definitions.

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Safety parameters.

To assess any potential toxicity of bortezomib with the dose and schedule of drug administration, specific laboratory parameters were tested in treated animals. No alterations in white blood cell count, hemoglobin, platelet count, or levels of aminotransferases, bilirubin, or creatinine were observed in bortezomib-treated animals as compared with vehicle-treated animals (Table 1). Arthritic rats developed anemia (mean ± SEM hematocrit 31.9 ± 3.4% in rats with AIA versus 44.3 ± 1.6% in normal rats [P = 0.03]; hemoglobin 9.5 ± 0.9 gm/dl in rats with AIA versus 14.2 ± 0.5 gm/dl in normal rats [P = 0.02]). Anemia improved in arthritic rats after bortezomib treatment, although not significantly (hematocrit 35.5 ± 1.3%; hemoglobin 11.0 ± 0.6 gm/dl [P = 0.5 for hematocrit and 0.4 for hemoglobin, versus rats with AIA]).

Table 1. Laboratory values in the normal rats, untreated rats with AIA, and bortezomib-treated rats*
 Normal ratsRats with AIABortezomib-treated rats
  • *

    Values are the mean ± SEM. AIA = adjuvant-induced arthritis; WBC = white blood cell; Hgb = hemoglobin; Hct = hematocrit; AST = aspartate aminotransferase; ALT = alanine aminotransferase; BUN = blood urea nitrogen.

  • P < 0.05 versus normal rats.

WBC count,/mm34,500 ± 1,4559,760 ± 1,92310,840 ± 1,917
Hgb, gm/dl14.2 ± 0.59.5 ± 0.911 ± 0.6
Hct, %44.3 ± 1.631.9 ± 3.435.5 ± 1.3
Platelet count, × 1,000/μl373 ± 103483 ± 74438 ± 102
AST, IU/liter63 ± 175 ± 16.684 ± 20
ALT, IU/liter47 ± 5.941 ± 3.7543 ± 2.2
Bilirubin, mg/dl0.6 ± 0.20.1 ± 0.00060.1 ± 0.01
BUN, mg/dl44 ± 5.531 ± 0.8533 ± 2.6
Creatinine, mg/dl0.4 ± 0.0050.4 ± 0.0350.3 ± 0.01

Alterations in the CD4+ cell numbers in PB and spleen of animals treated with bortezomib.

T cells and macrophages play a central role in the pathogenesis and progression of RA. In PB, the numbers of CD4+ cells were significantly higher in arthritic compared with normal rats and were further increased by bortezomib treatment (mean ± SEM CD4+ cells [× 104/ml] 577 ± 77 in arthritic rats versus 268 ± 34 in normal rats [P < 0.001] and versus 821 ± 163 in bortezomib-treated rats [P < 0.001]). Arthritic rats also had increased numbers of circulating CD8+ cells and CD11b+ cells compared with normal animals (mean ± SEM CD8+ cells [× 104/ml] 382 ± 63 in arthritic rats versus 146 ± 40 in normal rats [P < 0.001]; CD11b+ cells [× 104/ml] 210 ± 76 in arthritic rats versus 81 ± 25 in normal rats [P < 0.001]), whereas bortezomib treatment did not significantly alter their absolute numbers in PB (Figure 5B).

Conversely, there was a significant reduction in the splenic content of CD4+, CD8+, and CD11b+ cells in arthritic versus normal animals, and bortezomib treatment increased the number of splenic CD4+ cells without altering the CD8+ or CD11b+ numbers (mean ± SEM CD4+ cells [× 104/ml] 1,742 ± 362 in arthritic rats versus 3,127 ± 884 in normal rats [P < 0.001] and versus 2,608 ± 621 in treated rats [P < 0.001]; CD8+ cells [× 104/ml] 1,207 ± 301 in arthritic rats versus 2,623 ± 679 in normal rats [P < 0.001] and versus 1,237 ± 315 in treated rats; CD11b+ [× 104/ml] 480 ± 174 in arthritic rats versus 1,700 ± 489 in normal rats [P < 0.001] and versus 284 ± 145 in treated rats) (Figure 5C).

Down-regulation of the increased expression of Toll-like receptors (TLRs) in PB and in cultured FLS treated with bortezomib.

To investigate whether bortezomib also affects the expression of TLR-2, TLR-3, and TLR-4, PB and cultured FLS from normal, arthritic, and bortezomib-treated rats were tested by real-time RT-PCR. Increased expression of all TLRs was found in the blood of rats with established disease (day 31 postimmunization) but not in rats with earlier disease (day 16 postimmunization), as compared with normal animals. TLRs were down-regulated both in blood and in cultured FLS from bortezomib-treated rats that were killed 31 days after CFA injection and 4 days after the last dose of bortezomib (P ≤ 0.005) (Figure 5D).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

RA is a chronic, systemic, inflammatory autoimmune disease associated with significant comorbidity for which no cure currently exists (21). Biologic agents, such as the TNF inhibitors (22, 23) and the anti-CD20+ or the anti–IL-6 receptor monoclonal antibodies (24–26), have increased the therapeutic options for patients with RA; however, >50% of patients treated with a TNF inhibitor do not meet the American College of Rheumatology 50% improvement criteria (22, 27), and many develop secondary failure or drug resistance (28). Although such patients may exhibit disease response to alternative biologic agents, the definitive role of these agents in treating this difficult-to-cure disease has yet to be defined in randomized prospective trials comparing the available therapeutic options. Given the lack of an optimal treatment in poor responders and the increased risk of opportunistic infections and malignancies with biologic agents (29, 30), there is an unmet need for the development of novel therapies with different modes of action and a favorable safety and tolerability profile.

Bortezomib is the first-in-class proteasome inhibitor available for human use in multiple myeloma (1–3). RA potentially represents an important clinical opportunity for bortezomib use because the drug targets multiple pathways through ubiquitin/proteasome inhibition, and rather than interrupting a single cytokine pathway, it may affect RA in a pleiotropic manner. The first evidence that a proteasome inhibitor, PS-341, exerts an antiinflammatory effect was shown in streptococcal cell wall–induced polyarthritis (31), and recently, attenuation of collagen-induced arthritis in bortezomib-treated mice was reported (32). In the present study, we showed that bortezomib, through antiproliferative and proapoptotic effects in splenocytes and FLS, targeted 2 critical cell populations involved in the development of autoimmunity and inflammation in RA. In vitro, bortezomib significantly inhibited the proliferation of and promoted apoptosis of splenocytes and FLS, reduced the invasiveness of FLS from rats with AIA, and modified the pattern of cytokine secretion in cultured splenocytes from rats with AIA.

An alteration in the homeostatic balance between synovial cell proliferation and apoptosis may result in either increased cell proliferation or insufficient apoptosis (33, 34), which contribute to synovial hyperplasia. In the present study, the loss of homeostasis was mainly due to excessive FLS proliferation in the arthritic synovium, which could not be compensated for by the rates of cell death. Bortezomib, by significantly decreasing proliferation and promoting apoptosis of FLS from rats with AIA, probably restored joint homeostasis. Consistent with the results of previous studies showing that sensitivity to bortezomib is markedly higher in activated T cells than in resting T cells (35), the proliferation of normal resting splenocytes was not impaired by bortezomib of the lowest concentrations, whereas its proapoptotic effect was stronger in FLS from rats with AIA compared with normal FLS; it was thus confirmed that activated cells are more vulnerable to bortezomib-induced growth inhibition and cell death.

In vivo, bortezomib markedly ameliorated AIA even when administered during an advanced disease phase. Joints from bortezomib-treated rats showed limited histologic damage and inflammation. Reduced synovial vessel density was also observed, either as a direct antiangiogenetic effect of bortezomib or as an epiphenomenon due to the altered metabolic needs resulting from decreased synovial hyperplasia and inflammation. In addition, after bortezomib treatment, there was an obvious bone healing effect in rats with AIA, as demonstrated by CT imaging and histologic absence of bone loss. The notion of an anabolic effect on bone formation is also supported by previous studies demonstrating that bortezomib, through NF-κB inhibition, induces the differentiation of mesenchymal stem/progenitor cells into osteoblasts (36), increases the serum levels of bone formation markers in multiple myeloma patients (37), and builds new bone mass (38).

TLRs and their signaling pathways are currently being validated as potential therapeutic targets for inflammatory joint disease (39). TLRs are primarily involved in the innate immune response through recognition of conserved pathogen-associated molecular patterns (40) or host-derived “danger signals” produced during tissue injury or inflammation (41). TLR ligands have been found in the joints of patients with RA (42, 43). TLR-2, TLR-3, and TLR-4 signaling pathways in synovial tissue are activated during the disease process (44, 45), and deletion of TLR-4 in a mouse model of arthritis conferred protection against disease (46). Bortezomib significantly down-regulated the expression of TLR-2, TLR-3, and TLR-4 in PB or cultured FLS from treated animals, indicating a previously unrecognized drug action and suggesting that bortezomib, through a global NF-κB blockade, may also target TLRs and their signaling pathways.

Invasive RA FLS bear a tumor-like phenotype which may stem from their exposure to the inflammatory milieu (47) or arise from the impairment of repair mechanisms in the arthritic joints. This highly invasive phenotype is probably maintained by continuous NF-κB activation, which prevents the differentiation of RA FLS into multilineage pathways (48). In our study, bortezomib, by inhibiting NF-κB, may have blocked the differentiation arrest and allowed the bone marrow mesenchymal stem cells, which are recruited to the inflamed joints, to differentiate into osteoblasts, chondrocytes, or normal FLS and repair the joint. Therefore, in addition to its antiinflammatory and bone healing function, bortezomib may also affect arthritis by modulating stem cell biology.

The clinical application of NF-κB inhibitors could theoretically be associated with severe side effects due to the global inhibition of NF-κB, which is also involved in normal cell physiology controlling immune responses (49). The treatment paradigm of bortezomib in multiple myeloma suggests that bortezomib has a well-established and acceptable safety and tolerability profile (50). However, peripheral neuropathy, thrombocytopenia, and viral infections are commonly seen in multiple myeloma patients treated with bortezomib. At the intraperitoneal dose of 0.25 mg/kg 2 days/week for 2 weeks that was tested in our study, no abnormal laboratory values were obtained. However, given the narrow therapeutic window observed in treated animals with AIA, the cytotoxicity of the drug at high concentrations, and the need for repeated treatment in chronic diseases such as RA, safety concerns may be raised, especially if doses higher than the standard doses used in multiple myeloma will be needed for efficacy in this setting. We converted the therapeutic dose of 0.25 mg/kg in rats to a human dose equivalent of 1.5 mg/m2 intraperitoneally (www.fda.gov/cder/onctool/animalquery.cfm), which is similar to the standard intravenous bortezomib dose (1.3 mg/m2) used for multiple myeloma treatment. This implies that a similar dose and treatment schedule as used in multiple myeloma may be effective and have acceptable toxicity in a clinical application of the drug in RA.

Bortezomib seems to affect AIA in a pleiotropic manner by targeting inflammation, cell proliferation, apoptosis, bone disease, and TLR expression. Bortezomib potentially represents an attractive therapeutic intervention in inflammatory conditions and a highly promising agent in the treatment of RA, that is worth exploring in a clinical setting.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Yannaki had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Yannaki, Papadopoulou, Kaloyannidis, Anagnostopoulos.

Acquisition of data. Yannaki, Papadopoulou, Athanasiou, Paraskeva, Bougiouklis, Palladas.

Analysis and interpretation of data. Yannaki, Papadopoulou, Yiangou.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES