Distribution of extracellular matrix macromolecules in the vestibular nuclei and cerebellum of the frog, Rana esculenta.

The axons of transected and re-apposed vestibulocochlear nerve of the frog, in contrast to mammalian species, regenerate and establish functional contacts within their original termination areas of the vestibular nuclear complex and the cerebellum. The lack of regenerative capability of the mammalian central nervous system (CNS) is partially attributed to various extracellular matrix (ECM) molecules, such as chondroitin sulfate proteoglycans (CSPG) and tenascin-R (TN-R), which exert inhibition on axon regeneration. In contrast to these molecules, hyaluronan (HA) was reported to be permissive for CNS regeneration. Using histochemical and immunohistochemical methods, we investigated the distribution pattern of these molecules in the medial (MVN), lateral (LVN), superior and descending vestibular nuclei and the cerebellum of the frog and detected regional differences in the organization of the ECM. In the vestibular nuclear complex, pericellular condensation of the ECM, the perineuronal nets (PNNs) were recognizable in the LVN and MVN and were positive only for HA. The neuropil of the vestibular nuclei showed either a diffuse appearance with varying intensity of reactions, or dots and ring-like structures, which may represent the perinodal ECM of the vestibular fibers. In the cerebellum, indistinct PNNs that were only labeled for HA were present in the granular layer. Our findings suggest that the HA-rich, but CSPG and TN-R-free PNNs may be associated with the high degree of plasticity and regenerative potential of the amphibian vestibular system.

Okadaic acid-induced inhibition of protein phosphatase 2A enhances chondrogenesis in chicken limb bud micromass cell cultures.

The role of major cellular serine/threonine-specific protein phosphatases, protein phosphatase 1 and 2A, was investigated during chicken cartilage differentiation under in vitro conditions. Activity of protein phosphatase 2A decreased parallel to differentiation of chondrogenic cells, whereas activity of protein phosphatase 1 remained unchanged as assayed in the supernatants of the homogenised chicken limb bud micromass cell cultures. When okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A was applied in 20 nM concentration for 4 h during the second and third culturing days, it significantly increased the size of metachromatic cartilage areas measured in 6-day-old colonies. Following okadaic acid treatments, a significant inhibition in the activity of protein phosphatase 2A was found, while the activity of protein phosphatase 1 was unaffected as measured an days 2 and 3. TRITC-phalloidin labelling demonstrated that okadaic acid disorganised actin filaments and induced rounding of chondrogenic cells. This deterioration of actin filaments was reversible. Electron microscopy and biochemical analysis of colonies revealed that the ultrastructure and major components of cartilage matrix remained unchanged under the effect of okadaic acid. Okadaic acid-treatment applied to cultures containing predominantly differentiated chondrocytes (after day 4) did not influence the cartilage formation. 3H-thymidine and bromodeoxyuridine incorporation-assays demonstrated enhanced cell proliferation in the okadaic acid-treated colonies compared to that of the untreated ones. Our results indicate, for the first time, that protein phosphatase 2A is involved in the regulation of chondrogenesis. Inhibition of protein phosphatase 2A with okadaic acid may result in increased chondrogenesis via modulation of proliferation and cytoskeletal organisation, as well as via alteration of protein kinase A-signaling pathway of the chondrogenic cells.

Type X collagen in human enamel development: a possible role in mineralization.

Although type X collagen is one of the key molecules in endochondral ossification, no data are available on whether it is present in dental structures when mineralization is proceeding. We therefore monitored the appearance of type X collagen in tooth germs of human samples ranging in gestational age from 17-week-old fetuses to 9-week-old newborn. Using immunohistochemistry, ELISA techniques, and Western blotting, we show that type X collagen is present in human tooth germ during enamel maturation. Intense immunohistochemical staining for collagen type X was observed in the enamel and in the apical parts of secretory ameloblast at the bell stage when the dentine and enamel matrix were already under formation. The odontoblasts, the dentine, and the pulp were not stained. In the early (9-week) postnatal stage, the staining for collagen type X in the enamel matrix was diminished, and only a very weak signal could be detected in the secretory ameloblasts. A positive reaction for collagen type X was also observed in ELISA assay of extracts obtained from human embryonic enamel and hypertrophic cartilage samples. The Western blot analysis of the enamel demonstrated that size of the molecule detected by MoAb X53 is characteristic of the type X collagen. This correlates well with our immunohistochemical findings. Based on these data, we propose that type X collagen is one of the candidate molecules present in the enamel matrix that might be involved in mineralization of the enamel.

Complex pattern of Th1 and Th2 activation with a preferential increase of autoreactive Th1 cells in BALB/c mice with proteoglycan (aggrecan)-induced arthritis.

The central role of CD4+ T cells and the balance between T helper (Th) subpopulations in the pathogenesis of autoimmune diseases have been extensively studied. Proteoglycan (aggrecan)-induced arthritis (PGIA) is a murine model for rheumatoid arthritis (RA), which is characterized by a Th1 dominance at the onset of the disease. In addition to CD4+ T cells, antigen-presenting B cells and autoantibodies seem to play an important role in the development and regulation of PGIA. To identify proteoglycan-specific CD4+ T cell subsets and Th1- and Th2-supported antibody isotypes during the progression of PGIA, spleen cells of proteoglycan-immunized BALB/c mice were harvested at different times of immunization, and at different stages of the disease, and their cytokine production and antigen-specific antibody isotype profiles were determined by enzyme-linked immunospot (ELISPOT) assays. Both Th1 and Th2 cytokine-producing cells, with the predominance of IL-4/IL-5-secreting cells, were detected during the prearthritic stage, and a shift toward a Th1 dominance was observed at the time of onset of arthritis. Tissue homogenates of acutely inflamed joints contained significantly higher levels of interferon-gamma than IL-4. The prearthritic period and both the acute and chronic phases of joint inflammation were characterized by IgG1 dominance in the sera and this correlated with the number of IgG1-secreting B cells in the spleen. However, the ratio of autoreactive IgG1/IgG2a-secreting cells decreased in arthritic animals. These results indicate the activation and possible regulatory roles of both Th1 and Th2 subsets in the autoimmune process, with the necessity of a relative increase of autoreactive Th1 cells for the induction of joint inflammation.

A proteoglycan (aggrecan)-specific T cell hybridoma induces arthritis in BALB/c mice.

Aggrecan, the high buoyant density cartilage proteoglycan (PG), has been shown to induce progressive polyarthritis and ankylosing spondylitis in genetically susceptible BALB/c mice. To further characterize the nature of the autopathogenic effector T cells operating in these mice and to determine the region(s) of the PG molecule recognized by these T cells, we generated PG-specific T cell hybridomas from arthritic mice. One of the PG-specific T cell hybridomas (5/4E8), when injected into naive irradiated BALB/c mice, was capable of inducing clinical and histopathologic signs of arthritis. Massive swelling and redness of the paws dominated the clinical picture. A reactive synovial cell proliferation, the accumulation of hybridoma and inflammatory cells in the enlarged joint space, the loss of PG from the superficial layer of the articular cartilage, and the erosion of articular surface were identical histopathologic signs to those found either in primary or adoptive transfer of PG-induced arthritis. The PG-specific and arthritogenic T cell hybridoma (5/4E8) expressed TCR-alpha beta + (V beta 4), CD4+, and CD8- phenotypes and belonged to the Th1 subset, as the cells secreted IL-2 and IFN-gamma, but not IL-4 upon PG stimulation, and the response was MHC class II (I-Ad)-restricted. These observations provide direct evidence that PG-specific Th cells play crucial roles in autoimmune arthritic processes.

Presentation of cartilage proteoglycan to a T cell hybridoma derived from a mouse with proteoglycan-induced arthritis.

Immunization of BALB/c mice with human fetal cartilage proteoglycan (PG) produces progressive polyarthritis, and T cells play key roles in the development of the disease. To gain an understanding of how PG is presented to autoreactive T cells by synovial antigen-presenting cells (APC), we examined the abilities of various syngeneic APC in presenting PG to a specific T cell hybridoma 5/4E8, derived from a mouse with PG-induced arthritis. A20 B lymphoma cells and spleen cells were strong presenters of PG, but synoviocytes and P388D1 macrophages could only present PG effectively after stimulation with interferon-gamma (IFN-gamma). The IFN-gamma exerted its effect by up-regulating both MHC class II and intercellular adhesion molecule-1 (ICAM-1) expression by these cells as neutralizing antibodies to Ia, LFA-1 and ICAM-1 inhibited presentation. Our studies also showed that synoviocytes and spleen cells took up and processed PG more rapidly than the cell lines. Cysteine and serine protease-dependent antigen presentation of PG was blocked at 4 degrees C, 18 degrees C and by chloroquine treatment, indicating that presentation required active uptake and processing in an acidic compartment, probably in lysosomes. Also, keratan sulphate-depleted and cyanogen bromide (CNBr) and 2-nitro-5-thiocyanobenzoic acid (NTCB)-cleaved PG elicited stronger T cell responses, as they were more easily processed than the native molecule. Furthermore, CNBr-generated peptides were presented by fixed APC, indicating that core protein fragments of cartilage PG can be presented directly by APC in context with MHC class II molecules.

Effect of pregnancy on proteoglycan-induced progressive polyarthritis in BALB/c mice: remission of disease activity.

Proteoglycan-induced arthritis is a murine autoimmune model displaying many similarities to human rheumatoid arthritis and ankylosing spondylitis, as has been documented by clinical, immunological and histopathological studies. Since the onset of arthritis correlates with the serum antibody level to mouse cartilage proteoglycan (PG), it is believed that these autoreactive antibodies may play crucial roles in the pathological mechanisms of PG-induced arthritis. We have found that fertility in these PG-induced arthritic mice had been reduced but, unlike collagen-induced arthritis, had not been completely lost. Moreover, pregnancy had a beneficial effect upon the clinical symptoms with very little or no influence on serum antibody levels. Although fertility was retained and arthritic mothers delivered healthy offspring, the birth frequency was significantly less than in non-arthritic age-matched controls. Furthermore, the presence of anti-PG autoantibodies (predominantly IgG1 subclass) transmitted from arthritic mothers to infants transplacentally and by milk during the lactation period did not render these offspring either resistant or more sensitive to subsequent induction of arthritis. Subsequent immunization of infants with 'arthritogenic' PG revealed an unaltered susceptibility to arthritis induction.