Francesco Grassi

IFN-γ stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation

T cell–produced cytokines play a pivotal role in the bone loss caused by inflammation, infection, and estrogen deficiency. IFN-γ is a major product of activated T helper cells that can function as a pro- or antiresorptive cytokine, but the reason why IFN-γ has variable effects in bone is unknown. Here we show that IFN-γ blunts osteoclast formation through direct targeting of osteoclast precursors but indirectly stimulates osteoclast formation and promotes bone resorption by stimulating antigen-dependent T cell activation and T cell secretion of the osteoclastogenic factors RANKL and TNF-α. Analysis of the in vivo effects of IFN-γ in 3 mouse models of bone loss — ovariectomy, LPS injection, and inflammation via silencing of TGF-β signaling in T cells — reveals that the net effect of IFN-γ in these conditions is that of stimulating bone resorption and bone loss. In summary, IFN-γ has both direct anti-osteoclastogenic and indirect pro-osteoclastogenic properties in vivo. Under conditions of estrogen deficiency, infection, and inflammation, the net balance of these 2 opposing forces is biased toward bone resorption. Inhibition of IFN-γ signaling may thus represent a novel strategy to simultaneously reduce inflammation and bone loss in common forms of osteoporosis.

Oxidative stress causes bone loss in estrogen-deficient mice through enhanced bone marrow dendritic cell activation

Increased production of tumor necrosis factor α (TNF) in the bone marrow (BM) in response to both oxidative stress and T cell activation contributes to the bone loss induced by estrogen deficiency, but it is presently unknown whether oxidative stress causes bone loss through T cells. Here we show that ovariectomy causes an accumulation in the BM of reactive oxygen species, which leads to increased production of TNF by activated T cells through up-regulation of the costimulatory molecule CD80 on dendritic cells. Accordingly, bone loss is prevented by treatment of ovariectomized mice with either antioxidants or CTLA4-Ig, an inhibitor of the CD80/CD28 pathway. In summary, reactive oxygen species accumulation in the BM is an upstream consequence of ovariectomy that leads to bone loss by activating T cells through enhanced activity of BM dendritic cells, and these findings suggest that the CD80/CD28 pathway may represent a therapeutic target for postmenopausal bone loss.

Anti‐Fas–induced apoptosis in chondrocytes reduced by hyaluronan: Evidence for CD44 and CD54 (intercellular adhesion molecule 1) involvement

OBJECTIVE To investigate the in vitro effect of therapeutic hyaluronan (HA) of 500-730 kd on anti-Fas-induced apoptosis of chondrocytes from osteoarthritis (OA) patients, and to assess its mechanism of action by analyzing the role of the 2 HA receptors, CD44 and CD54 (intercellular adhesion molecule 1 [ICAM-1]). METHODS Chondrocytes isolated from human OA knee cartilage were cultured and the effect of HA on both spontaneous and anti-Fas-induced apoptosis was evaluated. Apoptosis was analyzed by JAM test (for quantitative analysis of fragmented DNA), cell death detection immunoassay (for quantitative analysis of oligonucleosome), TUNEL assay, and electron microscopy. Blocking experiments with anti-CD44 and anti-CD54 alone or in combination were performed to investigate the HA mechanism of action. RESULTS Both quantitative tests demonstrated that anti-Fas significantly induced apoptosis of isolated OA chondrocytes. HA at 1,000 microg/ml significantly reduced the anti-Fas-induced apoptosis of chondrocytes but did not affect spontaneous chondrocyte apoptosis. These data were also confirmed by TUNEL staining and by electron microscopy morphologic evaluation. The antiapoptotic effects of HA on anti-FAS-induced chondrocyte apoptosis were significantly decreased by both anti-CD44 (mean +/- SD 57 +/- 12% inhibition) and anti-ICAM-1 (31 +/- 22% inhibition). The mixture of the 2 antibodies had an additive effect, since the rate of inhibition increased to 87 +/- 13%. CONCLUSION These data demonstrate that 500-730-kd HA exerts an antiapoptotic effect on anti-FAS-induced chondrocyte apoptosis by binding its specific receptors (CD44 and ICAM-1). Furthermore, this HA fraction may be able to slow down chondrocyte apoptosis in OA by regulating the processes of cartilage matrix degradation.

T Cells Potentiate PTH-Induced Cortical Bone Loss through CD40L Signaling

Parathyroid hormone (PTH) promotes bone catabolism by targeting bone marrow (BM) stromal cells (SCs) and their osteoblastic progeny. Here we show that a continuous infusion of PTH that mimics hyperparathyroidism fails to induce osteoclast formation, bone resorption, and cortical bone loss in mice lacking T cells. T cells provide proliferative and survival cues to SCs and sensitize SCs to PTH through CD40 ligand (CD40L), a surface molecule of activated T cells that induces CD40 signaling in SCs. As a result, deletion of T cells or T cell-expressed CD40L blunts the bone catabolic activity of PTH by decreasing bone marrow SC number, the receptor activator of nuclear factor-kappaB ligand (RANKL)/OSTEOPROTEGERN (OPG) ratio, and osteoclastogenic activity. Therefore, T cells play an essential permissive role in hyperparathyroidism as they influence SC proliferation, life span, and function through CD40L. T cell-SC crosstalk pathways may thus provide pharmacological targets for PTH-induced bone disease.

Human osteoblasts express functional CXC chemokine receptors 3 and 5: Activation by their ligands, CXCL10 and CXCL13, significantly induces alkaline phosphatase and β-N-acetylhexosaminidase release

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 β‐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.

CXCL12 (SDF‐1) and CXCL13 (BCA‐1) chemokines significantly induce proliferation and collagen type I expression in osteoblasts from osteoarthritis patients

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.

IL1β and TNFα differently modulate CXCL13 chemokine in stromal cells and osteoblasts isolated from osteoarthritis patients: Evidence of changes associated to cell maturation

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.

Recruitment and proliferation of T lymphocytes is supported by IFNγ- and TNFα-activated human osteoblasts: Involvement of CD54 (ICAM-1) and CD106 (VCAM-1) adhesion molecules and CXCR3 chemokine receptor

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, TNFα and IFNγ, 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 IFNγ, either alone or in combination with TNFα, significantly up‐regulates the expression of CD54 and CD106 and induces the expression and release of CXCL9, CXCL10, CXCL11 in OB. The supernatant of TNFα‐ and IFNγ‐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 TNFα‐ and IFNγ‐activated OB or by incubation with the supernatant of TNFα‐ and IFNγ‐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. J. Cell. Physiol. 198: 388–398, 2004© 2003 Wiley‐Liss, Inc.

Inhibition of antigen presentation and T cell costimulation blocks PTH-induced bone loss

T cells are required for continuous parathyroid hormone (cPTH) treatment to induce bone loss as they sensitize stromal cells to PTH through CD40 ligand (CD40L), a surface molecule of activated T cells. Since CD40L expression is a feature of activated T cells, we investigated whether antigen (Ag)‐mediated T cell activation is required for PTH to exert its catabolic activity. We report that inhibition of Ag presentation through silencing of either class I or class II MHC‐T cell receptor (TCR) interaction prevents the cortical bone loss induced by in vivo cPTH treatment. We also show that the bone loss and the stimulation of bone resorption induced by cPTH treatment are prevented by CTLA4‐Ig, an inhibitor of T cell costimulation approved for the treatment of rheumatoid arthritis. Since inhibition of antigen‐driven T cell activation by blockade of either TCR signaling or T cell costimulation is sufficient to silence the catabolic activity of cPTH, antigen‐presenting cells and T lymphocyte interactions therefore play a critical role in the mechanism of action of PTH.

Sodium hydrosulfide inhibits the differentiation of osteoclast progenitor cells via NRF2-dependent mechanism.

Hydrogen sulfide (H2S), which recently emerged as a potent regulator of tissues and organs, is broadly produced in mammalian cells but whether it can regulate bone cell function is still elusive. The main objective of this study was to establish the role of H2S in the regulation of human osteoclast differentiation and function. Sodium hydrosulfide (NaHS), a common H2S-donor, was administered in vitro to CD11b+ human monocytes, the pool of circulating osteoclasts precursors which are critically involved in osteoclast development and function in bone. NaHS dose-dependently decreased human osteoclast differentiation at concentrations which did not induce toxicity. The inhibition of human osteoclast differentiation was associated with a down-regulation in RANKL-dependent intracellular ROS levels in human pre-osteoclasts cells. Furthermore, NaHS up-regulated NRF2 protein expression, its nuclear translocation, and the transcription of the two key downstream antioxidant genes Peroxiredoxin-1 and NAD(P)H dehydrogenase quinone 1, suggesting that NRF2 activation may inhibit human osteoclast differentiation by activating a sustained antioxidant response in osteoclast progenitors; furthermore, NRF2 activators Sulforaphane and Tert-butylhydroquinone inhibited in vitro human osteoclast differentiation. Moreover, silencing NRF2 in human pre-osteoclasts totally abolished NaHS-mediated inhibition of osteoclastogenesis, suggesting that NRF2 is essential to the inhibitory function of NaHS in osteoclast development. Finally, we found that NaHS also downregulated the RANKL/OPG mRNA ratio in human mesenchymal stem cells, the key osteoclast-supporting cells. Our results suggest that NaHS shows a potential therapeutical role in erosive diseases of bone by regulating both direct and indirect mechanisms controlling the differentiation of circulating osteoclasts precursors.