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. 2011 Feb;15(2):270-9.
doi: 10.1111/j.1582-4934.2009.00984.x.

Evidence for the prevention of enthesitis in HLA-B27/hβ(2)m transgenic rats treated with a monoclonal antibody against TNF-α

Anna Franca Milia  1 , Lidia Ibba-Manneschi, Mirko Manetti, Gemma Benelli, Sergio Generini, Luca Messerini, Marco Matucci-Cerinic
Affiliations

Evidence for the prevention of enthesitis in HLA-B27/hβ(2)m transgenic rats treated with a monoclonal antibody against TNF-α

Anna Franca Milia et al. J Cell Mol Med. 2011 Feb.

Abstract

Transgenic rats with high expression of HLA-B27 and human β(2) -microglobulin (B27TR) develop a multisystem inflammatory disease resembling human inflammatory bowel disease (IBD) and spondyloarthropaties (SpA). Tumour necrosis factor α (TNF-α) has a crucial role in sustaining chronic inflammation in the gut and joints. The aim of this work was to evaluate whether TNF-α blockade could prevent or reduce the inflammation of peripheral joints in B27TR. A first group of 9-week-old B27TR received an anti-TNF-α monoclonal antibody (mAb) or an isotypic IgG2a,k up to the age of 18 weeks. An untreated group was monitored up to the age of 18 weeks and then randomly assigned to a 9-week treatment with anti-TNF-α mAb or IgG2a,k. Each rat was monitored for clinical IBD and peripheral joint manifestations. After sacrifice the colon and hind paws were examined for macroscopical and microscopical pathological changes. Early TNF-α blockade prevented, and late treatment improved IBD signs in B27TR. Erythema, oedema, inflammatory infiltrate close to the tendons and enthesis, proliferating chondrocyte-like cells, signs of new endochondral bone ossification and bone erosion were observed in peripheral joints of four out of six IgG2a,k-treated B27TR, both at 18 and 27 weeks. Immunopositivity for phosphorylated Smad1/5/8 indicated that the process of joint remodelling was activated in B27TR. Some entheses showed chondroid nodules. Anti-TNF-α treatment reduced inflammation and preserved the enthesis organization in most animals. Occasional and transient erythema and oedema were still present in three of six of the late anti-TNF-α-treated animals. Smad1/5/8 signalling was not inhibited by late anti-TNF-α treatment. In B27TR, articular involvement follows IBD onset and develops at entheses. Early TNF-α blockade prevents the onset of IBD and consequently the development of enthesitis in peripheral joints in the B27TR model of human SpA.

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Figures

Fig 1
Fig 1
Macroscopic and microscopic features of joint disease in IgG2a,k-treated HLA-B27 transgenic rats (B27TR). (A) Oedema and erythema in the hind paw. (B)–(K) Representative histological sections of peripheral joints (B, E: haematoxylin and eosin staining, F: Goldner’s Masson trichrome staining, (G)–(K) toluidine blue staining). (B) Subcutaneous inflammatory infiltrate (asterisk) and oedema close to the enthesis and tendons in a 18-week-old rat. (C), (D) Representative microphotographs of immunostaining for CD3 and CD68 in the same rat showed in (B). (C) Immunohistochemistry showed that most of the infiltrating cells are CD3+ T cells; Inset: Higher magnification of CD3+ T cells. (D) Some CD68+ macrophages are present in the connective tissue. (E) Bone erosions (arrows) and inflammatory infiltrate (double asterisk) in a 18-week-old rat; Inset: Higher magnification of bone erosion areas. (F) Representative histological section of the peripheral joint of a 27-week-old rat; Inset: Higher magnification of the boxed area in F showing bone erosion with osteoclasts. (G) Reduced thickness of articular cartilage with hypertrophic chondrocytes, fibroblast-like cell proliferation (arrow) and irregular articular lining (double asterisk) in a 18-week-old rat. (H) Higher magnification of the articular cartilage shown in (G). Areas of superficial matrix pallor with necrosis and degeneration of chondrocytes are present. (I) Derangement of articular cartilage with proliferating chondrocyte-like cells and endochondral bone ossification in a 27-week-old rat. (J) Altered enthesis with proliferating chondrocyte-like cells and hypertrophic chondrocytes (arrow) in a 18-week-old rat. (K) Chondrocyte proliferation and endochondral bone ossification close to the tidemark (arrow) in a 27-week-old rat. Original magnification: (I) ×4; (F) ×10; (B–E), (G), (K): ×20; (J) ×40; (H) ×60; Insets: ×60.
Fig 3
Fig 3
Effects of anti-TNF-α mAb treatment on peripheral joint disease in HLA-B27 transgenic rats (B27TR). Results of histological score of inflammation, cartilage, enthesis and bone changes in 18-week-old B27TR (A) and 27-week-old B27TR (B) are shown. Each dot represents the value for an individual rat. Horizontal lines indicate the group medians. P < 0.05: anti-TNF-α-treated B27TR versus IgG2a,k-treated B27TR. n.s., not significant.
Fig 4
Fig 4
Representative immunostaining for TNF-α in the peripheral joints of HLA-B27 transgenic rats (B27TR). (A), (B) 18-week-old IgG2a,k-treated B27TR. (B) is a higher magnification view of boxed area in (A). Immunopositive chondrocytes in the fibrocartilaginous point of entheseal attachment (B) and in the proliferating zone at the border between bone and cartilage (arrow). (C) 27-week-old IgG2a,k-treated B27TR: immunopositive proliferating chondrocytes in the articular cartilage. (D) 18-week-old anti-TNF-α treated B27TR: absence of TNF-α immunopositivity in the cartilage. Original magnification: (A), (C), (D) × 20; (B) × 60.
Fig 5
Fig 5
Representative immunohistochemical results of phosphorylated Smad1/5/8 in the peripheral joints of HLA-B27 transgenic rats (B27TR). (A), (B) 18-week-old IgG2a,k treated B27TR. (B) is a higher magnification view of boxed area in (A). Smad1/5/8 signalling is detected in spindle-shaped entheseal fibroblast-like cells (black arrowhead), inflammatory cells (white arrowhead), and in round chondroblast-like cells (black arrows) at entheseal level. (C), (D) 27-week-old IgG2a,k treated B27TR. (D) is a higher magnification view of boxed area in (C). Proliferating (white arrowhead) and prehypertrophic (black arrow) chondrocyte-like cells show phosphorylated Smad1/5/8-immunopositivity in the fibrocartilage at the point of entheseal attachment. Hypertrophic chondrocytes are negative (black arrowhead). (E), (F) 27-week-old IgG2a,k treated B27TR. (F) is a higher magnification view of boxed area in (E). Immunopositive proliferating chondrocytes are evident in the articular cartilage (black arrow). (G), (H) 18-week-old anti-TNF-α treated B27TR. Absence of phosphorylated Smad1/5/8 immunopositivity in the articular cartilage (G) and at the entheseal level (H). (I), (J) 27-week-old anti-TNF-α treated B27TR. (J) is a higher magnification view of boxed area in (I). Proliferating chondrocytes in the fibrocartilaginous point of entheseal attachment show strong immunopositivity for phosphorylated Smad1/5/8 (black arrow). Original magnification: (G) ×10; (A, C, E, H, I) ×20; (B, D, F, J): ×40.
Fig 2
Fig 2
Microscopic features of disease in anti-TNF-α-treated HLA-B27 transgenic rats (B27TR). (A)–(D) Early anti-TNF-α treatment. (A) Histological section of colon in early-treated B27TR (haematoxylin and eosin staining). (B), (C) Representative histological sections of peripheral joint, with well preserved articular components. (D) Normal organization of the enthesis, with regular tidemarks and aligned chondrocyte rows towards the ligament. (B–D: toluidine blue staining). Original magnification: (A, C) ×10; (B) ×4; (D) ×40. (E)–(H) Late anti-TNF-α treatment. (E) Histological section of colon in late-treated B27TR (haematoxylin and eosin staining). (F)–(H) The histopathological joint features of three out of six late anti-TNF-α treated B27TR are shown. The other three late treated-B27TR showed no significant joint alterations as in the early treated group (B–D). (F) Areas of superficial matrix pallor, with hypertrophic chondrocytes and undulating articular surface. (G) Irregular tidemark between fibrocartilage and bone and endochondral bone ossification (arrow). (H) Bone erosion at the enthesis (arrow). [(F–H) toluidine blue staining]. Original magnification: (E–G) ×20; (H) ×40.
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