C.A. Poole

Ultrastructural, tomographic and confocal imaging of the chondrocyte primary cilium in situ

Hyaline cartilage chondrocytes express one primary cilium per cell, but its function remains unknown. We examined the ultrastructure of chick embryo sternal chondrocyte cilia and their interaction with extracellular matrix molecules by transmission electron microscopy (TEM) and, for the first time, double‐tilt electron tomography. Ciliary bending was also examined by confocal immunohistochemistry. Tomography and TEM showed the ciliary axoneme to interdigitate amongst collagen fibres and condensed proteoglycans. TEM also revealed the presence of electron‐opaque particles in the proximal axoneme which may represent intraciliary‐transport (ICT) particles. We observed a wide range of ciliary bending patterns. Some conformed to a heavy elastica model associated with shear stress. Others were acutely deformed, suggesting ciliary deflection by collagen fibres and proteoglycans with which the cilia make contact. We conclude that mechanical forces transmitted through these matrix macromolecules bend the primary cilium, identifying it as a potential mechanosensor involved in skeletal patterning and growth.

Mechanical loading modulates chondrocyte primary cilia incidence and length

The pathways by which chondrocytes of articular cartilage sense their mechanical environment are unclear. Compelling structural evidence suggests that chondrocyte primary cilia are mechanosensory organelles. This study used a 3D agarose culture model to examine the effect of compressive strain on chondrocyte cilia. Chondrocyte/agarose constructs were subjected to cyclic compression (0–15%; 1 Hz) for 0.5–48 h. Additional constructs were compressed for 48 h and allowed to recover for 72 h in uncompressed free‐swelling conditions. Incidence and length of cilia labelled with anti‐acetylated α‐tubulin were examined using confocal microscopy. In free‐swelling chondrocytes, these parameters increased progressively, but showed a significant decrease following 24 or 48 h compression. A 72 h recovery partially reversed this effect. The reduced cilia incidence and length were not due to increased cell division. We therefore propose that control of primary cilia length is an adaptive signalling mechanism in response to varying levels and duration of mechanical loads during joint activity.

Chondrocyte deformation within mechanically and enzymatically extracted chondrons compressed in agarose

Within articular cartilage, the chondron microenvironment will influence chondrocyte behaviour and response to loading. Chondrons were extracted from intact cartilage using either mechanical homogenisation (MC) or enzymatic digestion (EC) and cell and matrix morphology in unstrained and compressed agarose constructs was examined. Isolated chondrocytes (IC) were used for comparison. Immunolocalisation of type VI collagen and keratan sulphate revealed differences in the structure of the pericellular microenvironment such that MC most closely resembled chondrons in situ. The unstrained cell diameters of IC and EC were larger than MC at day 1 and increased significantly over a 7 day culture period. In contrast, cell diameters for MC remained constant. Compression of constructs at day 1 resulted in cell deformation for IC and EC but not MC. The two chondron extraction methods yielded chondrons of differing matrix morphology and associated differences in cell size and cellular response to load. The results indicate that the pericellular microenvironment of MC initially possessed a greater mechanical integrity than that of EC. Although these differences may be reduced with time in culture, characterisation of mechanically isolated chondrons suggests that the stiffness of the chondrons in situ may be greater than previous estimates.

The distribution of type VI collagen in the developing tissues of the bovine femoral head

Type VI collagen appears central to the maintenance of tissue integrity. In adult articular cartilage, type VI collagen is preferentially localised in the chondron where it may be involved in cell attachment. In actively remodelling developing cartilage, the distribution is less certain. We have used confocal immunohistochemistry and in situ hybridisation to investigate type VI collagen distribution in third trimester bovine proximal femoral epiphyses. In general, type VI collagen immunofluorescence was concentrated in the chondrocyte pericellular matrix, with staining intensity strongest in regions which persist to maturity and weakest in regions that remodel during development. Type VI collagen was also present in cartilage canals. In the growth plate and around the secondary centre of ossification, the intensity of type VI collagen stain rapidly decreased with chondrocyte maturation and was absent at hypertrophy, except where canal branches penetrated the growth plate and stain was retained around the adjacent chondrocytes. In situ hybridisation confirmed the presence of type VI collagen mRNA in cartilage canal mesenchymal cells but the signal was low in chondrocytes, suggesting minimal levels of synthesis and turnover. The results are consistent with a role for type VI collagen in stabilising the extracellular matrix during development.

Inducible nitric oxide synthase in pancreatic islets of the non-obese diabetic mouse: a light and confocal microscopical study of its ontogeny, co-localization and up-regulation following cytokine administration

Nitric oxide has been shown to mediate beta-cell destruction in rodent islets exposed to interleukin 1β in culture. The inhibitory effect is potentiated by tumour necrosis factor-α and interferon- γ. Cytokine stimulation leads to gene transcription and translation of inducible nitric oxide synthase, the biosynthetic enzyme of nitric oxide. In the non-obese diabetic mouse, progressive invasion of pancreatic islets by immune cells may lead to local production of inflammatory cytokines, resulting in inducible nitric oxide synthase expression within the islets. In this study, the ontogeny of this enzyme and its cellular expression were examined in pancreatic sections of female non- obese diabetic mice by double-label immunofluorescence. Light and confocal microscopy were employed to study the up-regulation, co- localization and immunocytoplasmic distribution of the enzyme in female non-obese diabetic and Swiss mice following cytokine treatment. From day 40 to day 220 a small number of beta-cells and a proportion of macrophages, usually in peri-islet and exocrine areas, expressed the enzyme. At onset of diabetes, an increasing number of macrophages within and surrounding the islets were positive for the enzyme. Treatment of day 60 female non-obese diabetic mice with interleukin 1β alone and in combination with tumour necrosis factor-α and/or interferon- γ resulted in a significant influx of macrophages into the pancreas, while this was lower in female Swiss mice treated similarly. Cytokine administration led to intense but sometimes eccentric immunocytoplasmic labelling for the enzyme in a considerable proportion of macrophages and beta-cells. Macrophages positive for inducible nitric oxide synthase were located in peri- and intra-islet areas, being distal and adjacent to enzyme-positive and negative beta-cells. Treatment with tumour necrosis factor-α and/or interferon-γ did not lead to enzyme up-regulation. These results show that in the non-obese diabetic mouse there is low and sustained expression of islet inducible nitric oxide synthase in the prediabetic period, which is followed by an increase around onset. However, treatment of female non-obese diabetic and Swiss mice with interleukin- 1β, alone or together with tumour necrosis factor-α and/or interferon-γ leads to a marked expression of this enzyme within macrophages and beta-cells

In vitro culture of enzymatically isolated chondrons: a possible model for the initiation of osteoarthritis.

The aim of this study was to assess whether enzymatically isolated chondrons from normal adult articular cartilage could be used as a model for the onset of osteoarthritis, by comparison with mechanically extracted chondrons from osteoarthritic cartilage. Enzymatically isolated chondrons (EC) were cultured for 4 weeks in alginate beads and agarose gel constructs. Samples were collected at days 1 and 2, and weekly thereafter. Samples were immunolabelled for types II and VI collagen, keratan sulphate and fibronectin and imaged using confocal microscopy. Mechanically extracted chondrons (MC) were isolated, immunohistochemically stained for type VI collagen and examined by confocal microscopy. In culture, EC showed the following characteristics: swelling of the chondron capsule, cell division within the capsule and remodelling of the pericellular microenvironment. This was followed by chondrocyte migration through gaps in the chondron capsule. Four types of cell clusters formed over time in both alginate beads and agarose constructs. Cells within clusters exhibited quite distinct morphologies and also differed in their patterns of matrix deposition. These differences in behaviour may be due to the origin of the chondrocytes in the intact tissue. The behaviour of EC in culture paralleled the range of morphologies observed in MC, which presented as single and double chondrons and large chondron clusters. This preliminary study indicates that EC in culture share similar structural characteristics with MC isolated from osteoarthritic cartilage, confirming that some processes that occur in osteoarthritis, such as pericellular remodelling, take place in EC cultures. The study of EC in culture may therefore provide an additional tool to investigate the mechanisms operating during the initial stages of osteoarthritis. Further investigation of specific osteoarthritic phenotype markers will, however, be required in order to validate the value of this model.

Temporal Relationship Between Immune Cell Influx and the Expression of Inducible Nitric Oxide Synthase, Interleukin-4 and Interferon-γ in Pancreatic Islets of NOD Mice Following Adoptive Transfer of Diabetic Spleen Cells

Beta cell destruction in NOD mice can be accelerated by adoptive transfer of diabetic spleen cells into irradiated adult NOD mice. Here mice receiving diabetic spleen cells were examined at days 0, 7, 14, 21 and at onset of diabetes for the resulting insulitis and the number of intra-islet CD4 and CD8 cells and macrophages. The progression of insulitis and the number of intra-islet CD4 and CD8 cells and macrophages were correlated with the expression and co-localization of inducible nitric oxide synthase, interferon-γ and interleukin-4 by dual-label light and confocal immunofluorescence microscopy. Diabetes developed in 7/8 mice by 27 days following cell transfer. The insulitis score increased slightly by day 7 but rose sharply at day 14 (p=0.001) and was maintained until diabetes. The mean number of intra-islet CD4 and CD8 cells and macrophages showed a similar trend to the insulitis scores and were present in almost equal numbers within the islets. Immunolabelling for inducible nitric oxide synthase was observed at day 7 in only some cells of a few islets but increased sharply from day 14. It was restricted to islets with insulitis and was co-localized in selective macrophages. Weak intra-islet interleukin-4 labelling was observed at days 7 and 14 but became more pronounced at day 21 and at onset of diabetes, being present in selective CD4 cells. Intra-islet labelling for interferon-γ was first observed at day 21, but became more intense at onset of diabetes and was co-localized in a proportion of macrophages. Both cytokines were expressed in islets with advanced insulitis. Interferon-γ staining was also observed within endothelial cells located in the exocrine pancreas. We conclude that transfer of diabetic spleen cells results in a rapid influx of CD4 and CD8 cells and macrophages within the pancreas of recipient mice. During the period of heightened insulitis, selective immune cells begin to express inducible nitric oxide synthase and the opposing cytokines, interferon-γ and interleukin-4. Expression of these molecules becomes more pronounced immediately prior to and during the onset of diabetes.

Dual-Label Immunohistochemical Study of Interleukin-4-and Interferon-γ-Expressing Cells within the Pancreas of the NOD Mouse During Disease Acceleration with Cyclophosphamide

Beta cell destruction has been shown to occur when rodent or human islets are exposed in vitro to inflammatory cytokines, such as interleukin-1β″ (IL-1β), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Other cytokines such as interleukin-4 (IL-4) or inter-leukin-10 (IL-10), when given to NOD mice, prevent insulin-dependent diabetes mellitus (IDDM). In this study, we have employed immunofluorescence histochemistry to study the expression of IFN-γ and IL-4 in the pancreas of female NOD mice at various time-points (days 0, 4, 7, 11 and at onset of diabetes) following disease acceleration with cyclophosphamide (Cy). Dual-label confocal and light microscopy were employed to determine the precise cellular sources of the two cytokines. IL-4 immunolabelling was observed in a few immune cells at days 0, 4, and 7 within the pancreatic islets but in larger numbers at day 11 and at onset of diabetes. The cytokine was co-localized predominantly in CD4 cells, while only a small minority of CD8 cells and macrophages also expressed IL-4. At days 0, 4, 7 and 11, weak to moderate immunolabelling for IL-4 was also observed in beta cells. In contrast, immunolabelling for IFN-γ within the islets was not observed until day 11 and this labelling persisted at onset of diabetes. It was immunolocalized in macrophages and to a lesser extent in CD4 cells. Only a few CD8 cells were immunopositive for IFN-γ. At day 11, a proportion of beta cells showed weak immunolabelling for IFN-γ. During the study period, immunolabelling for IFN-γ was also observed in a proportion of endothelial cells located in the intra-islet and exocrine regions of Cy and diluent-treated mice. From day 11 onwards, both the cytokines were observed in some of the peri-vascular regions. Our results demonstrate that during Cy-induced diabetes, there is increasing expression of both IL-4 and IFN-γ in specific immune cells within the inflamed islets in the late prediabetic stage and at onset of diabetes. Further studies are required to correlate our protein immunohistochemical findings with in situ cytokine gene expression and to determine whether there is a clear Thl cytokine protein bias at clinical onset of diabetes and immediately preceding it

An Immunohistochemical Study of Macrophage Influx and the Co-localization of Inducible Nitric Oxide Synthase in the Pancreas of Non-obese Diabetic (NOD) Mice During Disease Acceleration with Cyclophosphamide

Cyclophosphamide has been used to accelerate and synchronize diabetes in non-obese diabetic (NOD) mice. It was injected to 70-day-old female NOD mice and its effect on the progression of insulitis studied at days 0, 4, 7, 11 and at onset of diabetes. Pancreatic sections were also examined for the influx of CD4 and CD8 T cells and macrophages following immunofluorescence staining. The kinetics of macrophage immunoreactive cells in the exocrine and intra-islet areas were also investigated. Light and confocal microscopy were employed to examine the expression and co-localization of inducible nitric oxide synthase following dual- and triple-label immunofluorescence histochemistry. After cyclophosphamide administration, the severity of insulitis remained similar from days 0 to 4 but began to rise at day 7 and markedly by day 11 and at onset of diabetes. At these two later stages, the insulitis scores were close to 100% while in age-matched control groups the insulitis scores were considerably lower. Immunohistochemical staining showed increasing numbers of CD4 and CD8 T cell subsets and macrophages within the islets and in exocrine, sinusoidal and peri-vascular regions. At onset of diabetes, several islets contained prominent clusters of macrophage immunoreactive cells. Macrophage influx into the islets increased sharply from day 7 (mean number per islet: 119±54 SEM), peaked at day 11 (mean number per islet: 228±42), and then declined at onset of diabetes (mean number per islet: 148±49). Several cells with immunolabelling for inducible nitric oxide synthase were detectable from day 7 onwards until the onset of diabetes. Dual- and triple-label immunohistochemistry showed that a significant proportion of macrophages and only a few beta cells contained the enzyme. Macrophages positive for the enzyme were located as clusters or occasionally contiguously, in the peri-islet and intra-islet areas but rarely in the exocrine region. Islets with minimal distribution of macrophages in the peri-islet areas were not positive for inducible nitric oxide synthase. Beta cells positive for the enzyme were observed in islets with significant macrophage infiltration in locations close to macrophages. The present results show that cyclophosphamide administration to female NOD mice results in a rapid influx of CD4 and CD8 cells and macrophages. The marked up-regulation of inducible nitric oxide synthase in a selective proportion of macrophages, within the islets, immediately preceding and during the onset of diabetes suggests that nitric oxide released by islet macrophages may be an important molecular mediator of beta cell destruction in this accelerated model of insulin-dependent diabetes mellitus.

THE CHONDROCYTE PRIMARY CILIUM – A PURINERGIC MECHANORECEPTOR?

Purpose: Mechanical loading is essential for the health and homeostasis of articular cartilage and may be utilised within a tissue engineering context to promote the production of a functional repair tissue. Consequently there is much interest in elucidating the fundamental mechanotransduction pathways in chondrocytes. Previous studies have demonstrated the presence of primary cilia in articular chondrocytes and their interaction with integrins and the extracellular matrix. Furthermore, we have recently shown that in Tg737orpk mice that lack polaris, a protein involved in cilia assembly, chondrocytes have extremely short or missing cilia. These mice have defects in growth plate and developing articular cartilage as well as skeletal patterning abnormalities and stunted growth. This suggests that primary cilia are critical for the development and health of articular cartilage although the precise function of this fascinating organelle in cartilage has not been established. In various other cell types, primary cilia act as mechanoreceptors, such that cilia deflection initiates intracellular Ca2+ signaling as part of a mechanotransduction cascade. We have demonstrated that compression of chondrocytes also activates a Ca2+ signalling pathway, mediated by the release of ATP, which triggers an up-regulation of proteoglycan synthesis. However, the mechanism of ATP release and the involvement of the primary cilium are as yet unclear. Methods: The present study used immunofluorescence and confocal microscopy of bovine and human articular cartilage to examine the expression and co-localisation of primary cilia and connexin 43 hemichannels, which are known to act as mechanosensitive ATP release channels in other cell types. In addition, we also examined the expression of purine receptors through which extracellular ATP may trigger Ca2+ signalling Results: Within bovine articular cartilage, approximately 40-60% of chondrocytes exhibited primary cilia with a higher percentage of ciliated cells in the deep zone. All cells were found to express connexin 43 hemichannels which was confirmed by immunolabelling of both the intracellular and extracellular domains. Furthermore, at least 50% of the primary cilia were decorated with connexin 43 (Fig. 1).