Andrea Facchini

Flow cytometric analysis of intracellular chemokines in chondrocytes in vivo: constitutive expression and enhancement in osteoarthritis and rheumatoid arthritis

Chemokines play a key role in modulating leukocyte functions at sites of inflammation. To assess chondrocyte contribution to the chemotactic environment of inflamed joints the intracellular content of CC and CXC chemokines was investigated. IL‐8, GROα, MCP‐1, RANTES, MIP‐1α and MIP‐1β expression was evaluated by flow cytometric analysis and RT‐PCR in chondrocytes isolated from cartilage specimens obtained from patients with osteoarthritis and rheumatoid arthritis and multiorgan donors as normal controls. All the chemokines except RANTES were found in normal chondrocytes, with different degrees of staining intensity. In osteoarthritis and rheumatoid arthritis patients, an enhancement of IL‐8, GROα, MIP‐1α and MIP‐1β was observed.

Vitamin D, thyroid hormones and muscle mass influence natural killer (NK) innate immunity in healthy nonagenarians and centenarians

Increasing evidence has demonstrated that the immune system closely interacts with other physiological systems, whose communications are mediated by circulating cytokines and hormones. The aim of our study was to test whether the number and cytolytic activity of NK cells in a group of relatively healthy Italian nonagenarians and centenarians were affected by the modifications of endocrine, metabolic and functional parameters that occur during ageing. Because of the extreme age of the study population, a cross‐sectional analysis was performed. This study revealed that the group of oldest subjects with the highest number of NK cells and the best preserved cytolytic function also presented a preserved metabolism of thyroid hormones and vitamin D and integrity of muscle mass. In fact, the NK cell number and/or cytolytic activity of healthy subjects > 90 years old was positively associated with serum levels of vitamin D, while T3, FT4, i‐PTH hormones and lean body mass were associated only with NK cell number. In conclusion, our results stress the paramount importance of nutritional evaluation in the clinical assessment of elderly people.

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.

Osteoblasts and stromal cells isolated from femora in rheumatoid arthritis (RA) and osteoarthritis (OA) patients express IL-11, leukaemia inhibitory factor and oncostatin M

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‐1β and tumour necrosis factor‐alpha (TNF‐α), 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‐1β and TNF‐α 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.

IL-17 enhances the susceptibility of U-2 OS osteosarcoma cells to NK cell lysis.

We investigated the effect of the proinflammatory cytokine interleukin 17 (IL‐17) on the lysis of osteosarcoma cells by human NK cells. NK cells and U‐2 OS, MG‐63, HOS osteosarcoma cell lines express the IL‐17 receptor, the highest amount being found on U‐2 OS. Pre‐incubation of NK cells with IL‐17 did not affect the cytotoxicity against osteosarcomas, that was increased when U‐2 OS were pre‐incubated with IL‐17. In IL‐17 treated U‐2 OS osteosarcoma cells FACS analysis demonstrated an increased expression of fibronectin among the panel of adhesion molecules assayed, and the treatment with anti‐fibronectin antibodies decreased the NK cytotoxicity. The comparison between interferon gamma (IFN‐γ) treated and IFN‐γ/IL‐17‐treated U‐2 OS showed a decreased susceptibility to NK lysis associated with a reduced expression of CD49f on U‐2 OS treated with IFN‐γ/IL‐17. IL‐17 appears to be a modulator of NK adhesion molecules on U‐2 OS cells but antagonizes with IFN‐γ on NK lysis.

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.

Human osteosarcoma cell susceptibility to natural killer cell lysis depends on CD54 and increases after TNFα incubation

Osteosarcoma cell lines vary widely in their susceptibility to natural killer (NK) cell lysis in vitro although it is still unclear why this occurs. In this study we investigated the expression of some cell adhesion molecules on osteosarcomas to determine which of these can modify the susceptibility to NK lysis and we also attempted to modulate the cytolytic susceptibility of these targets with TNFα. We found that osteosarcoma lysis induced by NK cells correlates with different expression of the CD54 adhesion molecule on osteosarcomas and the increased susceptibility after TNFα treatment mostly depends on the expression of CD54 molecules on target cells.

EPITOPE SPECIFICITY OF TH0/TH2 CD4+ T-LYMPHOCYTE CLONES INDUCED BY VACCINATION WITH RHBSAG VACCINE

MARIA CRISTINA HONORATI,* ,‡ PAOLO DOLZANI,* ERMINIA MARIANI,* ,‡ ANNA PIACENTINI,* GINA LISIGNOLI,* CARLO FERRARI, and ANDREA FACCHINI* ,‡ *Laboratorio di Immunologia e Genetica, Istituto di Ricerca Codivilla Putti –Istituti Ortopedici Rizzoli, Bologna; Dipartimento di Medicina Interna e Gastroenterologia, Universita di Bologna, Bologna; Cattedra di Malattie Infettive, Universita di Parma, Parma; and Divisione di Malattie Infettive e Immunopatologia Virale, Azienda Ospedaliera di Parma, Parma, Italy