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Comparative Study
. 2005;7(5):R927-37.
doi: 10.1186/ar1766. Epub 2005 Jun 6.

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants in vitro

Duncan E Crombie  1 , Muhammed Turer, Beltzane Biurrun Zuasti, Bayden Wood, Don McNaughton, Kutty Selva Nandakumar, Rikard Holmdahl, Marie-Paule Van Damme, Merrill J Rowley
Affiliations
Comparative Study

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants in vitro

Duncan E Crombie et al. Arthritis Res Ther. 2005.

Abstract

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6). Penetration of cartilage by mAb was determined by immunofluorescence on frozen sections and correlated with changes to the extracellular matrix and chondrocytes by morphometric analysis of sections stained with toluidine blue. The effects of mAbs on matrix components were examined by Fourier transform infrared microspectroscopy (FTIRM). A possible role of Fc-binding was investigated using F(ab)2 from CIIC1. All three mAbs to CII penetrated the cartilage explants and CIIC1 and M2139, but not CIIF4, had adverse effects that included proteoglycan loss correlating with mAb penetration, the later development in cultures of an abnormal superficial cellular layer, and an increased proportion of empty chondrons. FTIRM showed depletion and denaturation of CII at the explant surface in the presence of CIIC1 or M2139, which paralleled proteoglycan loss. The effects of F(ab)2 were greater than those of intact CIIC1. Our results indicate that mAbs to CII can adversely affect preformed cartilage, and that the specific epitope on CII recognised by the mAb determines both arthritogenicity in vivo and adverse effects in vitro. We conclude that antibodies to CII can have pathogenic effects that are independent of inflammatory mediators or Fc-binding.

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Figures

Figure 1
Figure 1
Immunofluorescence showing the penetration of the three anti-CII antibodies: (a) CIIF4, (b) CIIC1 and (c) M2139. The area of colour indicates antibody binding. (d) The control mAb (GAD6) shows no binding to the cartilage.
Figure 2
Figure 2
A toluidine blue stained sections of cartilage. (a) Cartilage cultured for 7 days shows an evenly stained matrix with typical rounded chondrocytes. Sections of cartilage incubated for (b) 7 days and (c) 14 days with M2139 show abnormal matrix morphology with the loss of toluidine blue, the development of a cellular layer at the surface and the development of hypertrophic chondrocytes.
Figure 3
Figure 3
Differences in the loss of proteoglycan and chondrocyte between cultures incubated with different mABs. (a) Loss of toluidine blue staining between cultures incubated with CIIF4 (white) and cultures incubated with CIIC1 (light grey) and M2139 (dark grey) over the course of 21 days. (b) The number of empty chondrons expressed as a percentage of the total number of chondrons, indicating the loss of chondrocyte from the extracellular matrix. The columns represent the mean of each measurement and error bars indicate 1 standard deviation. The asterix represents p < 0.05.
Figure 4
Figure 4
FTIRM spectra of the major cartilage components CII, proteoglycan and hyaluronan. (a) Typical spectra for CII, crude proteoglycan extract and hyaluronan. (b) An artificial spectrum that resembles normal articular cartilage generated by combining appropriate proportional amounts of the spectra of CII (55%), crude proteoglycan extract (40%) and hyaluronan (5%). The amide 1 peak from 1600–1700 cm-1 represents the total protein content, the triplet of peaks from 1200–1300 cm-1 are characteristic of the spectrum of collagen, and the peaks in the region 960–1175 cm-1 result from sugars in the proteoglycans and hyaluronan.
Figure 5
Figure 5
Distribution of proteoglycans in the cultured explants. (a) Toluidine blue stained sections cultured for 14 days with GAD6. (b) Chemical map derived using FTIRM showing the proteoglycan region (960–1175 cm-1). The chemical maps show the distribution and relative concentrations of proteoglycans; the least concentrated areas are shown as blue and the most concentrated areas that are shown as red. (c) The spectra shown are the mean of 10 measurements taken from either the central areas (red line) or near the surface of the tissue (blue line). The error bars represent 1 standard deviation at those points in the spectra. The amide 1 region, which represents the total protein content of the tissue, is from 1600–1700 cm-1.
Figure 6
Figure 6
Distribution of proteoglycans in the explants cultured with CIIF4 or M2139. Toluidine blue stained sections cultured for 14 days with (a) CIIF4 or (d) M2139 are shown alongside (b, e) chemical maps showing proteoglycan distribution and (c, f) FTIRM spectra from the central areas (red line) and near the surface of the tissue (blue line). The error bars represent 1 standard deviation at those points in the spectra.
Figure 7
Figure 7
Distribution of proteoglycans in the explants cultured with CII-C1 or F(ab)2 from CII-C1. Toluidine blue stained sections cultured for 14 days with (a) CII-C1 or (d) F(ab)2 are shown alongside (b, e) chemical maps showing proteoglycan distribution and (c, f) FTIRM spectra from the central areas (red line) and near the surface of the tissue (blue line). Note that the proteoglycan levels are lower and the amide 1 peak has shifted across the whole of the F(ab)2 treated tissue. The error bars represent 1 standard deviation at those points in the spectra.
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