In previous work, the correlation of IGFBP-3 levels with OA was examined closely by determining the relationship between levels of the binding protein and individual OA scores11(Abstract)
. It could be demonstrated that while the relationship was weak, there was a statistically significant correlation between IGFBP-3 and OA score. There was also a molar dominance of IGF-I over IGFBP-3 in many of the OA samples, irrespective of score11
. This led us to ask whether different sub anatomical pools contain high pockets of IGFBP-3 that may be diluted or obscured in the tissue extracts, but which may play different roles during OA.
Since there are a number of reports indicating that IGFBP-3 is found within the cell in various cell lines, the question of whether some of the IGFBP-3 is present in chondrocytes was raised. We first addressed this question by preparing fresh chondrocytes and lysing them after minimal culture (overnight in spinner culture). This short incubation period was designed to allow cell recovery from proteolytic matrix excision, while minimizing pericellular protein accumulation. shows an IGF Ligand Blot in which chondrocyte lysates were run alongside cartilage extracts and the membranes probed with a 125I-IGF-II ligand. It can be clearly seen that the cell lysates displayed a very strong positive signal coincident with IGFBP-3. The cell lysate of the 10-04D sample can be directly compared to the tissue extract from the same sample (): the lysate showed a much stronger signal even though equal amounts of protein were added to each lane. The more extensive dilution of the whole tissue extracts with matrix protein may have contributed to this. However, the finding that both cell lysates displayed strong signals for IGFBP-3 was consistent with the possibility that a significant portion of the IGFBP-3 in cartilage was cell-associated. We then needed to rule out the possibility that cellular expression was a consequence of the preparative procedures (release of chondrocytes from the matrix by proteolysis) and/or the short incubation period of the cells prior to lysis.
125IGF-II Western Ligand Blots of Cell Lysates Compared to Total Tissue Extracts
To explore the cellular localization of IGFBP-3 in intact cartilage we carried out immunohistochemistry of representative slices from normal human cartilage (Normal, Mankin grade 1; IGFBP-3 content =6 ng/mg protein), from intermediate (score 6; 15 ng IGFBP-3/mg protein) and advanced OA grades (score 9; 17 ng IGFBP-3/mg protein). clearly shows the presence of cell-associated IGFBP-3 in slices of normal human cartilage (panels A and B) and in sections of cartilage from a donor with intermediate OA (panels C and D). In addition, panels A and B show that cartilage from a donor with severe OA also shows strong cellular immunostain, which includes the cloned cells. Matrix stain was noted in all sections. In the normal cartilage, staining was seen as a thin layer on the top zone of the section (, and ). In the OA samples, there was more staining, which extended deeper into the section (Figure and ) but remained mostly confined to ~the top half of the section.
Immunohistochemistry for IGFBP-3 of Samples with Normal and Intermediate Stage Osteoarthritis
Immunohistochemistry for IGFBP-3 of Samples with Advanced Osteoarthritis
Immunohistochemistry of Nuclei for IGFBP-3 in Normal Cartilage
In all samples at all OA stages, >90% of the cells were clearly positive for IGFBP-3 content. Importantly, many of the cells in all samples had a strong nuclear IGFBP-3 signal. This can be appreciated best in high-resolution photographs of normal cartilage shown in panels A and B, which show intense but discreet immunoreactivity associated with the nucleus. In addition, many of the cells had a web-like IGFBP-3 pattern outside of the nucleus (). Chondrocytes in the deep zone of the normal cartilage specimens were negative while the OA samples showed a positive cellular signal in this zone (compare to ). Controls were prepared for each of the samples either without anti-IGFBP-3 or with anti-BP-3 previously saturated with IGFBP-3 antigen. The negative antibody controls were negative for cells in all the samples (see for example), and the antigen-blocked antibody controls consistently showed very reduced cell immunoreactivity (e.g. ). The matrix stain was also blocked or largely reduced in all control samples. However, it needs to be pointed out that the intermediate OA sample () had particularly intense matrix stain in the top zone (partially shown in the top of ) and diffuse stain remained in the antigen-blocked controls (not shown).
We then asked whether the IGF-I ligand showed a similar distribution in sections from the same samples examined by IGFBP-3 IHC. shows the close association of IGF-I with the chondrocyte in the normal (panel A) and severe OA (panel B) cartilages. Panel C shows a fuller view of the cartilage with intermediate OA; there is more intense IGF-I stain in the upper half of the cartilage in both cells and matrix. This general distribution pattern was also observed in the normal and severe OA samples. Panel D shows the negative control performed without antibody with greatly diminished stain in both the matrix and the cell compartments. Similarly, antigen-blocked controls showed greatly reduced immunostain. In contrast to the IGFBP-3 distribution, IGF-I did not show a dominant association with the nucleus.
Immunohistochemical staining for IGF-I
The apparent nuclear localization of IGFBP-3 deserved closer scrutiny. Frozen sections of 19-year old human cartilage from an ankle joint were examined. shows the immunofluorescent IGFBP-3 stain in the left side panels (A & C) and the DAPI stain in the right side panels (B & D) for adjacent sections. The coincidence between the staining patterns can be seen, indicative of immunoreactive IGFBP-3 in association with the nucleus. shows the overlay of the two images from panels C & D. Extra nuclear stain can also be appreciated. Negative controls (without primary antibody) were blank (not shown).
Fluorescent immunomicroscopy of IGFBP-3 and DAPI Stained Nuclei in Young Human Cartilage
We then prepared thin sections and used immunogold-labeling techniques in order to ascertain if the immunoreactivity associated with the nucleus was intra-nuclear. The tissue source for these studies was a human knee from an OA arthroplasty; the histological OA grade (7.5) indicated that this was an intermediate-to-Severe OA sample. , panels A and C shows thin sections (1 μm) of chondrocytes with stained nuclei (arrows) in the transitional zone and the superficial zone respectively, examined by light microscopy. , panel E shows the immunogold TEM, which further supports the intra-nuclear location of IGFBP-3.
Immunogold and Immunohistochemical Staining on Thin Sections of Human Cartilage