Intra-articular

Blom AB, van Lent PL, Libregts S, Holthuysen AE, van der Kraan PM, van Rooijen N, van den Berg W. Crucial role of macrophages in matrix metalloproteinase–mediated cartilage destruction during experimental osteoarthritis : Involvement of matrix metalloproteinase 3. Arthritis & Rheumatism. 2007;56(1):147–57.

Abstract

Objective: To explore the involvement of synovial macrophages in early cartilage damage in osteoarthritis (OA), and to identify the role of matrix metalloproteinase 3 (MMP-3) in the pathology of early and late OA.

Methods: The role of synovial macrophages in MMP-mediated damage in OA was studied by depleting synovial macrophages prior to elicitation of a collagenase-induced instability model of OA. The expression of MMP in synovium and cartilage was monitored using TaqMan analysis. In spontaneous and induced OA, cartilage pathology was scored in MMP-3–knockout mice and control mice, by histologic assessment and VDIPEN staining.

Results: On day 14 following induction of OA, MMP-mediated neoepitopes were detected in cartilage from mice with mild experimental OA (mean
SD positively stained surface area 20
3.2%). Remarkably, by depleting synovial macrophages prior to induction of OA, the generation of MMP-induced neoepitopes was largely prevented (mean
SD positively stained surface area 5
1%; P< 0.001), indicating an important role for synovial macrophages in the occurrence of MMPmediated cartilage damage. We observed a strong decrease in MMP-3 and MMP-9 expression in synovial but not cartilage tissue in macrophage-depleted joints. Among 2-year-old mice, spontaneous OA–like changes in the lining layer were significantly decreased in MMP- 3–knockout mice compared with control mice. Even more striking was the 67% reduction in the occurrence of severe cartilage damage in MMP-3–knockout mice. In addition, MMP-mediated VDIPEN expression was significantly decreased, indicating reduced MMPmediated cartilage breakdown.

Conclusion: The results of this study prove that MMP-3 is involved in the generation of severe cartilage damage in murine OA. Synovial macrophages are crucial in early MMP activity and appear to mediate MMP production in synovium rather than cartilage. [custom_table]

Clodronate Concentration	Total Lipid Concentration	Lipid Composition	Lipid Mole %	Liposome Type	Control Liposomes
not specified	not specified	EPC/Chol	not specified	MLV	none

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Animal Description	Clodronate Dose	Dosing Method/Site	Target Phagocytes	Systemic Dosing?	Systemic Results
C57BL/6 mice2	not specified	intra-articular/knee	F4/80+ synovial lining MΦ	no	N/A

²Base strain. Other genetically altered or knockout strains were also used.

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Notes

1. Liposome prep method referenced—van Rooijen N, Sanders A, van den Berg TK. Apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine. Journal of Immunological Methods. 1996 Jun;193(1):93–9.
2. Animals were injected 1 week prior to induction of arthritis with collagenase.
3. No details of injection method or clodronate liposome dose were published, however referenced paper states that 6 µl (75 µg clodronate) were injected into the joint.

Results

1. F4/80+ MΦ were reduced from about 45% to 15% by day 14 and 50% to 15% by day 21 post-injection of clodronate liposomes (7 and 14 days post-induction of OA) by immunohistological analysis.
2. Depletion was not detected in the sublining.
3. Although the authors state that F4/80+ MΦ had begun to repopulate at 3 w post-injection of clodronate liposomes in a cited article, the depletion level had not changed in the current experiment.
4. F4/80+ MΦ depletion correlated with a significant down-regulation in MMP-3.
5. The authors further pursue in the possible roles of MMP’s in MMP knockout mice without the use of clodronate liposomes.

 

Barrera P, Blom A, van Lent PLEM, van Bloois L, Beijnen JH, van Rooijen N, de Waal Malefijt M, van de Putte L, Storm G, van den Berg W.
Synovial macrophage depletion with clodronate-containing liposomes in rheumatoid arthritis.
Arthritis & Rheumatism. 2000;43(9):1951–9.

[toggle_content title=”Abstract”]Objective: To assess whether intraarticular (IA) administration of clodronate liposomes results in local macrophage depletion in patients with rheumatoid arthritis (RA). Primary goals were to address both the immunohistologic and potential toxic effects of this approach. Moreover, the correlation between immunohistologic findings and clinical assessments of disease activity and cartilage damage were assessed.

Methods: An open study was conducted in consecutive RA patients who were scheduled for knee joint replacement in our department. Synovial biopsy tissue was obtained from the knee joint at 2 weeks before and at the time of surgery. This protocol was controlled for safety and immunohistologic concordance in 6 patients. One week before surgery, 10 patients received a single IA dose of clodronate liposomes. Staining of synovial tissue for cell markers (CD68, CD14, CD3, CD38) and adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1]) was assessed by 2 blinded observers. Local and systemic parameters of disease activity were measured before each intervention. Cartilage damage was scored using standard radiologic techniques at baseline and during surgery.

Results: A single IA dose of clodronate liposomes significantly reduced the number of CD68-positive cells (P = 0.005) and the expression of ICAM-1 and VCAM-1 in the synovial lining (P = 0.013 and P = 0.039, respectively). The intervention did not affect fibroblast-like synoviocytes, T cells, or plasma cells. No immunohistologic changes were observed in the control group. The procedure was well tolerated. The levels of ICAM-1 and VCAM-1 in the sublining layers correlated with the extent of macroscopic synovitis (P < 0.0005 and P < 0.005, respectively). The expression of ICAM-1 and CD14 in the sublining correlated with the levels of C-reactive protein (P < 0.0005 and P < 0.01, respectively). Cartilage destruction was correlated only with the expression of CD68 in the sublining (P = 0.02).

Conclusion: A single IA administration of clodronate liposomes leads to macrophage depletion and decreased expression of adhesion molecules in the synovial lining in patients with longstanding RA. The procedure is well tolerated, and its therapeutic potential is currently under investigation. The expression of adhesion molecules in the sublining layers reflects ongoing inflammation.[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]

Clodronate Concentration	Total Lipid Concentration	Lipid Composition	Lipid Mole %	Liposome Type	Control Liposomes
4-10 mg/ml	not specified	EPC/Chol	not specified	LUV, 120-160 nm	none

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Animal Description	Clodronate Dose	Dosing Method/Site	Target Phagocytes	Systemic Dosing?	Systemic Results
Humans, >18 yr, scheduled for knee joint prosthesis	160 ± 35 mg	intra-articular/knee	CD68+ synovial lining/sublining MΦ/synoviocytes	no	N/A

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1. Liposome prep method did not specify amount of EPC used, but appeared to be a printing error. Maintaining the 6:1 molar ratio typically employed by van Rooijen would have called for 1.1 g EPC per 98 mg chol which was specified in the text. If we assume final prep volume was 10 ml, then the total lipid concentration would have been ~115 mg/ml. EPC/chol MLV were extruded through 0.2, 0.1 and 0.05 µm filters. Only 60 mg/ml clodronate used for resuspension (van Rooijen method uses 250 mg/ml) and encapsulation efficiency reported at 10-14%. Using the 10 ml total prep volume assumption and 10% encapsulation efficiency, patients received ~27 ml injections. We have not previously seen this prep method and the assumptions may not have provided the correct injection volumes.
2. The authors stated that the dose was extrapolated from animal studies, but no further details were given.
3. Patients were treated 2 weeks prior to knee joint prosthetic surgery during which histolgical samples were collected and other observations recorded.
4. Study goal was to evaluate safety and tolerance.

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1. Median CD68+ cells in synovial lining reduced from 4 to 0.45 (immunohistological analysis).
2. Median CD68+ cells in synovial sublining reduced from 2.65 to 1.65, although authors speculate that the reduction was not due to liposomes entering the sublining.
3. VCAM-1 and ICAM-1 were significantly reduced in the synovial lining in treated patients which correlated with a reduction in synovitis and macroscopic swelling at the time of surgery.
4. CD14 and ICAM-1 correlated with CRP.
5. CD68 staining in the sublining correlated with cartilage damage.
6. In similarly designed animal studies, depleted macrophages were completely repopulated in 2-4 weeks post-treatment.
7. Patients reported no discomfort or pain upon clodronate liposome injection and no adverse reactions were noted.
8. Hisological analysis revealed substantial changes in the appearance of the synovial linings.

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