Search

To evaluate the role of CXC chemokines CXCL8 (IL8), CXCL10 (IP-10), CXCL12 (SDF-1), and CXCL13 (BCA-1) in bone remodeling, we analyzed their effects on osteoblasts (OBs) obtained from subchondral trabecular bone tissue of osteoarthritis (OA) and post-traumatic (PT) patients. The expression of CXC receptors/ligands (CXCR1/CXCL8, CXCR2/CXCL8, CXCR3/CXCL10, CXCR4/CXCL12, and CXCR5/CXCL13) was analyzed in cultured OBs by flow cytometry and immunocytochemistry. Functional assays on CXC chemokine-treated-OBs in the presence or absence of their specific inhibitors were performed to analyze cellular proliferation and the enzymatic response to chemokine activation. The expression of chemokine ligands/receptors was also confirmed in bone tissue samples by immunohistochemical analysis. Collagen type I and alkaline phosphatase mRNA expression were analyzed on CXCL12- and CXCL13-treated OBs by real-time PCR. OBs from both OA and PT patients expressed high levels of CXCR3 and CXCR5 and lower amounts of CXCR1 and CXCR4. CXCL12 and CXCL13, only in OBs from OA patients, induced a significant proliferation that was also confirmed by specific blocking experiments. Moreover, OBs from OA patients released a higher amount of CXCL13 than those of PT patients while no differences were found for CXCL12. In the remodeling area of bone tissue samples, immunohistochemical analysis confirmed that OBs expressed CXCL12/CXCR4 and CXCL13/CXCR5 both in OA and PT samples. CXCL12 and CXCL13 upregulated collagen type I mRNA expression in OBs from OA patients. These data suggest that CXCL12 and CXCL13 may directly modulate cellular proliferation and collagen type I in OA patients, so contributing to the remodeling process that occurs in the evolution of this disease., (Copyright 2005 Wiley-Liss, Inc.)

Bone homeostasis is regulated by cells at different stages of maturation that are influenced by soluble factors. The modulatory function of two pro-inflammatory cytokines, IL-1beta and TNF-alpha, on the expression of CXCL13 chemokine was evaluated in osteoblasts (OB) and bone marrow stromal cells (BMSC) from osteoarthritis (OA) and post-traumatic (PT) patients. In basal condition, CXCL13 production by both BMSC and OB was significantly higher in OA than in PT patients. IL1beta, significantly induced CXCL13 production in differentiated OB, both from OA and PT patients, but not in BMSC from both either group. TNFalpha reduced CXCL13 production only in BMSC from OA patients. The combination of IL1beta and TNFalpha increased CXCL13 production only in OB in the same amount as for IL-1beta alone. OB from OA released a higher amount of CXCL13 compared to PT in all conditions tested. CD121a (IL1 receptor type I) was highly expressed only by OB. Moreover, in bone tissue biopsies CXCL13 was expressed by mesenchymal and mononuclear cells. This study demonstrates that cells at different stages of maturation (BMSC and OB) and derived from physiological (PT) or pathological conditions (OA) respond in different ways to inflammatory stimuli. These data may contribute to understand the basic maturation processes of bone cells in old patients.

The mechanism by which osteoblasts (OB) interact and modulate the phenotype and proliferation of T lymphocytes during inflammation is not well known. The effects of two regulatory cytokines, TNFalpha and IFNgamma, on the expression of CD54 (ICAM-1) and CD106 (VCAM-1) adhesion molecules and the CXCR3 ligands (CXCL9, CXCL10, CXCL11), were assessed in a primary culture of human OB by real-time PCR, flow cytometry, and immunohistochemistry. In addition, we functionally evaluated the recruitment and proliferation of T lymphocytes grown with resting or stimulated OB. According to the present data IFNgamma, either alone or in combination with TNFalpha, significantly up-regulates the expression of CD54 and CD106 and induces the expression and release of CXCL9, CXCL10, CXCL11 in OB. The supernatant of TNFalpha- and IFNgamma-activated OB induces the recruitment of T lymphocytes more significantly than stimulation by CXCR3 ligands. T lymphocyte proliferation is significantly enhanced by direct contact with TNFalpha- and IFNgamma-activated OB or by incubation with the supernatant of TNFalpha- and IFNgamma-activated OB. Blocking experiments with anti-CD11a, anti-CD49d, anti-CXCR3, and Bordetella pertussis toxin demonstrate that adhesion molecules and the CXCR3 chemokine receptor play a key role in the proliferation of T lymphocytes. The present study demonstrates the involvement of adhesion molecules (CD11a and CD49d) and chemokine receptor (CXCR3) in the mechanism by which OB recruit, interact, and modulate T lymphocyte proliferation under inflammatory conditions., (Copyright 2003 Wiley-Liss, Inc.)

The expression and functional activity of CXC chemokine receptors were evaluated in human osteoblasts (OB) obtained post-trauma from old donors compared to very young donors. It was found that CXCR1 and CXCR4 were only expressed by old but not young donors' cells. In contrast, CXCR3 and CXCR5 were expressed by both young and old donors. We functionally evaluated CXCR3/CXCL10 and CXCR5/CXCL13 receptor/ligand pairs by analysing cell proliferation and the release of N-acetyl-beta-D-glucosaminidase (NAG), an enzyme that degrades glycosaminoglycans and hyaluronic acid. CXCL10 and CXCL13 induced a dose-dependent increase of cell proliferation in OB from young donors while cell proliferation of OB in old donors was not affected. By contrast, CXCL10 and CXCL13 induced a significantly higher NAG release in OB from old donors compared to young ones. These data demonstrate a significant age-dependent difference in the response of OB to CXCL10 and CXCL13 stimulation. These chemokines induce an inverse response of OB from old and young donors, which suggests a role of ageing in the modulation of cellular response of bone cells.

Osteoblasts (OBs) contribute to the maintenance of bone homeostasis and their activity can be influenced by immune cells localized in bone lacunae. We investigated the expression of the chemokine receptors in isolated human OBs by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry, and report a novel finding, namely, that OBs express high levels of CXC chemokine receptor 3 (CXCR3) and 5 (CXCR5). Functional assays to evaluate CXCR3 and CXCR5 demonstrated that their ligands-CXCL10 and CXCL13, respectively-significantly induce the release of beta-N-acetylhexosaminidase, an enzyme involved in endochondral ossification and bone remodeling able to degrade important extracellular matrix components. Alkaline phosphatase activity, a useful index of matrix formation was also up-regulated by CXCL10 and CXCL13. However, OB activation by these ligands does not affect OB proliferation. Both Bordetella pertussis toxin and neutralizing anti-CXCR3/anti-CXCR5 monoclonal antibodies block CXCL10 and CXCL13 induction, respectively. We also demonstrated the expression of CXCL10 and CXCL13 in human bone tissue biopsies. These results indicate that both CXCR3/CXCL10 and CXCR5/CXCL13 receptor-ligand pairs may play an important role in OB activity through the specific up-regulation of two enzymes, which are involved in the bone remodeling process. Moreover, our data suggest that OBs may play a role in the modulation of bone formation through the combined action of these two enzymes., (Copyright 2002 Wiley-Liss, Inc.)

In the present study we analyse chemokine expression in the remodelling of subchondral bone in arthritis patients. Trabecular bone biopsies were tested by immunohistochemistry to identify interleukin (IL)-8, GRO-alpha, MCP-1, RANTES, MIP-1alpha and MIP-1beta expression. Subsequently, we evaluated by immunoassay the effect of interferon (IFN)-gamma and IL-6 on chemokine production by osteoarthritis (OA), rheumatoid arthritis (RA) and post-traumatic (PT) patients' isolated osteoblasts (OB). OB constitutively produced in situ IL-8, GRO-alpha, MCP-1, RANTES and MIP-1alpha. MIP-1beta was positive only in mononuclear cells. In RA many of these chemokines were also produced by mononuclear cells. IFN-gamma significantly down-regulated IL-8 and up-regulated MCP-1 produced by OB from all patients tested, whereas it did not affect the other chemokines analysed. Moreover, IFN-gamma reduced IL-1beta-stimulated IL-8 production but significantly increased both MCP-1 and RANTES. Interestingly, IL-6 significantly downregulated IFN-gamma-induced MCP-1 production, that was significantly lower in OA compared to RA patients. OB expressed chemokines both in vivo and in vitro suggesting that these cells are primary effectors in the bone capable of regulating autocrine/paracrine circuits that affect bone remodelling in these diseases.

We investigated both in vitro and ex vivo the role of mature osteoblasts (OB) and bone marrow stromal cells (BMSC) in RA and OA by analysing the expression of the following IL-6-type cytokines: IL-11, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and IL-6. OB and BMSC were isolated from femora of RA, OA and post-traumatic (PT) patients, cultured in vitro in the presence or absence of IL-1beta and tumour necrosis factor-alpha (TNF-alpha), and assessed for the production and mRNA expression of IL-6-type cytokines. Trabecular bone biopsies were obtained from the inner portions of femoral heads and used for cytokine in situ immunostaining. Cultured OB and BMSC from different patients constitutively secreted IL-11 and IL-6 but not OSM. LIF was secreted only by BMSC, at very low levels. Interestingly, IL-11 basal production was significantly higher in BMSC than in OB in all three groups tested. IL-1beta and TNF-alpha strongly stimulated IL-6-type cytokine release (except for OSM) by both OB and BMSC. OSM was expressed only at mRNA levels in all groups studied. Cytokine immunostaining on bone biopsies confirmed the data obtained on cultured cells: IL-11, IL-6 and LIF proteins were detected both in mesenchymal (BMSC and OB) and mononuclear cells; OSM was found only in mononuclear cells. These data demonstrate that IL-6-type cytokines are constitutively expressed in the bone compartment in RA, OA and PT patients and can be secreted by bone cells at different stages of differentiation (BMSC and OB). This suggests that these cytokines may be involved in the mechanisms of bone remodelling in OA and RA.

Objective: To evaluate whether subchondral osteoblasts (OB) are involved in the production of cytokines and chemokines in rheumatic diseases., Methods: OB were isolated from subchondral bone of rheumatoid arthritis (RA), osteoarthritis (OA) and post-traumatic (PT) patients, cultured in vitro in the presence or absence of interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), and assessed for the production, immunolocalization, and mRNA expression of proinflammatory cytokines (IL-1alpha, IL-1beta, TNF-alpha) and alpha and beta chemokines [IL-8, growth related gene product (GRO-alpha), monocyte chemoattractant protein 1 (MCP-1), RANTES, and macrophage inflammatory proteins MIP-1alpha, MIP-1beta]., Results: Cultured OB from different patients did not release IL-1alpha, IL-1beta, or TNF-alpha, and constitutively secreted IL-8, GRO-alpha, and MCP-1, while RANTES, MIP-1alpha, MIP-1beta were undetectable or near the lower level of sensitivity of the immunoenzymatic assay. GRO-alpha was significantly higher in RA than in OA and PT patients. IL-1beta and TNF-alpha alone or in combination strongly stimulated chemokine release by OB. Only RANTES production was not increased by the combination of the 2 cytokines. IL-1alpha, IL-1beta, and TNF-alpha were expressed as cytoplasmic proteins and were not secreted by OB even after stimulation., Conclusion: OB from subchondral bone release chemokines that could be involved in the mechanisms that directly or indirectly cause bone remodelling and cartilage destruction.

Osteoblastic cells are in direct or indirect contact with lymphocytes and bone marrow cells that are the main targets of HIV-1. Therefore we analysed whether HIV-1 could infect these cells using three bone cell lines (HOS, MG-63, U2 OS) as a model. These cells were infected with HIV-1 (strain NL4-3) and the supernatants were harvested every day for 20 days for p24 antigen measured using an ELISA immunoassay. The DNA of infected cells was extracted at days 3, 9, and 12 and the PCR for gag gene was performed using Jurkat cell line as a negative control and ACH-2 cell line as a positive control. Our results demonstrated that HOS, MG-63 and U2 OS are not infected by HIV-1. These data were confirmed using PCR that is currently the most sensitive procedure available.