Sophie Roux

Expression of BAFF (BLyS) in T cells infiltrating labial salivary glands from patients with Sjögren's syndrome

Primary Sjögrens syndrome (pSS) is an autoimmune disorder characterized by lymphocytic infiltration of the salivary glands. Most of the infiltrating cells are T cells, but other features of the disease include polyclonal B‐cell activation, systemic production of autoantibodies, and increased risk of developing B‐cell non‐Hodgkins lymphoma. Recently, a new tumour necrosis factor, the B‐cell activating factor (BAFF; also known as BLyS), has been implicated in the polyclonal activation of B cells. Using immunohistochemistry, this study evaluated BAFF expression in labial salivary gland biopsies from 14 patients with pSS, 14 normal controls, and two patients with sarcoidosis. Labial salivary gland samples from seven patients with pSS, seven controls, and one patient with sarcoidosis were double‐stained using indirect immunofluorescence. RT‐PCR analysis was also performed on lip biopsy samples from two patients and two controls. In all 14 pSS specimens, infiltrating inflammatory cells strongly expressed BAFF protein, as did some ductal epithelial cells, but acinar cells were negative. Some B cells were present in the vicinity of the BAFF‐positive cells. In the 14 normal labial salivary glands, some ductal cells were moderately positive, but acinar cells were negative. In the labial salivary glands from the two patients with sarcoidosis, infiltrating lymphocytes were not stained. BAFF mRNA expression was confirmed by RT‐PCR in salivary glands from pSS patients. Double immunofluorescence revealed T cells and macrophages to be the main cell types expressing BAFF in salivary glands from pSS patients. In conclusion, BAFF may be expressed by T cells at the site of autoimmune damage and could play a role in the pathogenesis of pSS, particularly by triggering the activation of self‐antigen‐driven autoimmune B cells. Copyright

RANK (receptor activator of nuclear factor-kappaB) and RANKL expression in multiple myeloma.

Summary.  The new members of the tumour necrosis factor (TNF) receptor‐ligand family, receptor activator of nuclear factor‐κB ligand (RANKL) and its receptor RANK, play a crucial role in osteoclast differentiation and activation. An increased expression of RANKL and/or RANK may be involved in the excessive bone resorption observed in multiple myeloma (MM). We used immunohistochemistry to study RANK and RANKL expression in bone marrow (BM) biopsies obtained at diagnosis in 15 MM patients, six patients with monoclonal gammopathy of undetermined significance (MGUS) and 10 normal BM biopsies. Plasma cells were not labelled with anti‐RANKL or anti‐RANK antibodies. In all biopsies, RANKL was expressed in endosteal bone surface, around vessels and in cells characterized by cytoplasmic expansions. These last cells did not express CD45 and were vimentin positive, corresponding to bone marrow stromal cells. Numerous stromal cells expressed RANKL in MM and MGUS specimens, with a greater expression in MM than in MGUS. Very few cells were stained with anti‐RANKL in normal BM specimens. With the anti‐RANK antibody, small mononuclear cells in the bone microenvironment were positive and were identified as erythroblast cells. In conclusion, we showed that RANKL was expressed in reticular stromal cells, with a greater intensity in myeloma specimens. These results suggest that RANKL overexpressed by bone marrow stromal cells may contribute to the high rate of bone resorption observed in MM.

RANK (Receptor Activator of Nuclear Factor kappa B) and RANK Ligand Are Expressed in Giant Cell Tumors of Bone

In giant cell tumors of bone (GCTBs), the mesenchymal stromal cells are the neoplastic cells and induce recruitment and formation of osteoclasts (OCs). Studies on recently discovered members of the tumor necrosis factor receptor-ligand family have demonstrated a crucial role of RANKL (receptor activator of nuclear factor kappa B [RANK] ligand) expressed by osteoblast/stromal cells and of its receptor RANK expressed by OCs during OC differentiation and activation. OCs typically are present in large numbers in GCTBs, suggesting that these tumors may contain cells expressing factors that stimulate OC precursor recruitment and differentiation. We used immunohistochemical analysis to study RANKL and RANK expression in 5 GCTBs. Multinucleated cells and some mononuclear cells showed strong positive staining with anti-RANK antibodies; RANKL was present in a subset of mononuclear cells that did not express the hematopoietic lineage cell marker CD45, a feature that identified them as mesenchymal tumor cells. Our results suggest that RANKL expression may have a role in the pathogenesis of GCTBs and in the formation of the large OC population present in these tumors.

In vivo inhibition of osteoblastic metalloproteinases leads to increased trabecular bone mass.

Mice specifically overexpressing TIMP‐1 in osteoblasts have been generated to investigate the role of MMPs in bone in vivo. These mice displayed increased trabecular bone volume and decreased bone turnover. This model provides evidence of the role played by the MMPs in bone remodeling and balance.

Human cord blood monocytes undergo terminal osteoclast differentiation in vitro in the presence of culture medium conditioned by giant cell tumor of bone.

Osteoclasts (OCs), which form by fusion of hematopoietic precursor cells, are typically present in large numbers in giant cell tumors of bone (GCTBs). These tumors may, therefore, contain cells which secrete factors that stimulate recruitment and differentiation of OC precursors. Multinucleated cells resembling OCs also form in cultures of human cord blood monocytes (CBMs) stimulated by 1.25 dihydroxyvitamin D3, but these cells lack the ability to form bone resorption pits, the defining functional characteristic of mature OCs. CBMs may thus require additional stimulation to form OCs; we therefore investigated whether GCTBs are a source of such a stimulus. CBMs were stimulated in long term (21 day) culture by medium conditioned by explants of GCTBs; media collected within 15 days of explant (early‐CM) and after 15 days (late‐CM) were employed. We also cocultured CBMs with primary GCTB‐derived stromal cells as well as immortalized bone marrow stroma‐derived cells. CBMs stimulated by early‐CM formed resorption pits on cortical bone slices; however, stimulation by late‐CM resulted in virtually no resorption. Both early‐CM and late‐CM increased CBM proliferation, but not the proportion of vitronectin receptor positive or multinucleated cells. Coculture of CBMs with stromal cells of GCTBs or bone marrow did not result in bone resorption, although these stromal cells (most expressing alkaline phosphatase but progressively losing parathyroid hormone receptor expression) expressed mRNA for cytokines involved in OC differentiation, including macrophage‐CSF, granulocyte‐macrophage‐CSF, IL‐11, IL‐6, and stem cell factor. Our results indicate that CBMs are capable of terminal OC differentiation in vitro, a process requiring 1,25 dihydroxyvitamin D3 as well as diffusible factor(s) which can be derived from GCTB. Stromal cells of GCTB may produce such factors in vivo, but do not support OC differentiation in vitro, possibly through phenotypic instability in culture.

Indapamide, a Thiazide-Like Diuretic, Decreases Bone Resorption In Vitro

We recently showed that indapamide (IDP), a thiazide‐related diuretic, increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium. In the present study, we evaluated the in vitro effects of this diuretic on bone cells, as well as those of hydrochlorothiazide (HCTZ), the reference thiazide, and acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor. We showed that 10−4 M IDP and 10−4 M AZ, as well as 10−5 M pamidronate (APD), decreased bone resorption in organ cultures and in cocultures of osteoblast‐like cells and bone marrow cells in the presence of 10−8 M 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3]. We investigated the mechanism of this antiresorptive effect of IDP; IDP decreased osteoclast differentiation as the number of osteoclasts developing in coculture of marrow and osteoblast‐like cells was decreased markedly. We then investigated whether IDP affected osteoblast‐like cells because these cells are involved in the osteoclast differentiation. Indeed, IDP increased osteoblast‐like cell proliferation and alkaline phosphatase (ALP) expression. Nevertheless, it did not modify the colony‐stimulating factor 1 (CSF‐1) production by these cells. In addition, osteoblast‐like cells expressed the Na+/Cl− cotransporter that is necessary for the renal action of thiazide diuretics, but IDP inhibited bone resorption in mice lacking this cotransporter, so the inhibition of bone resorption and osteoclast differentiation did not involve this pathway. Thus, we hypothesized that IDP may act directly on cells of the osteoclast lineage. We observed that resorption pits produced by spleen cells cultured in the presence of soluble osteoclast differentiation factor (sODF) and CSF‐1 were decreased by 10−4 M IDP as well as 10−5 M APD. In conclusion, in vitro IDP increased osteoblast proliferation and decreased bone resorption at least in part by decreasing osteoclast differentiation via a direct effect on hematopoietic precursors.

Effects of Prostaglandins on Human Hematopoietic Osteoclast Precursors

The effect of prostaglandin E2 (PGE2) on osteoclast (OC) differentiation is unclear, either stimulator or inhibitor, depending on the in vitro system used. This probably reflects indirect mechanisms through intermediate cells. We have investigated the direct effect of PGE2 on human OC differentiation from cord blood monocytes (CBMs) in the absence of stromal cells. Macrophages and multinucleated cells (MNCs) resembling OCs form in cultures of CBMs stimulated by 1,25-dihydroxyvitamin D3. In the present study, CBMs were cultured for 3 weeks, as previously described, in the presence or absence of PGE2. The number of MNCs was significantly reduced in the presence of PGE2 as was the proliferation of cultured CBMs, assessed on day 7. Immunohistochemistry was performed to evaluate macrophage markers (CD11b and CD14) and OC marker (beta3-chain). PGE2 significantly increased the numbers of CD11b-positive and CD14-positive cells, whereas the number of beta3-chain-positive cells was significantly decreased. beta3-Chain, c-fos, and human calcitonin receptor (h-CTR) messenger RNA (mRNA) expressions were evaluated by reverse transcription-PCR with RNA extracted from cultured CBMs. In the presence of PGE2, expression of beta3-chain and c-fos mRNA was reduced from the first week of culture. h-CTR mRNA expression was also reduced, and only the h-CTR1 isoform was detected in the presence of PGE2. In addition, when PGE2 was added only during the last week of culture, when no CBM proliferation occurred, the number of CD11b- and beta3-positive cells was unchanged compared to that in the control culture, as were the proportion of MNCs, the fusion index, and the expression of c-fos mRNA. In conclusion, our results suggest that PGE2 has an inhibitory effect on human OC differentiation from CBMs, possibly by reducing precursor proliferation in these cultures. We also hypothesize that PGE2 may reduce OC differentiation by increasing the proportion of precursor cells that differentiate into macrophages. In addition, this may be the result of inhibition of the c-fos expression in CBMs.

Type-I 15-hydroxyprostaglandin dehydrogenase: role in macrophage/osteoclast differentiation.

Vitamin D3 acts on several cellular processes, including cellular growth and differentiation. Osteoclasts (OCs), are multinucleated bone-resorbing cells formed by fusion of mononuclear cells of hematopoietic origin. OCs share committed hematopoietic progenitors with monocytes and are suspected to be bone-adaptated macrophages (Udagawa et al., 1990). Studying the genes, which could be transcriptionaly regulated by vitamin D3, during the differentiation of hematopoietic precursors of OCs, we showed that type-I 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH [EC.1.1.1.141]) (Ensor et al., 1990) gene activation was one of the major regulatory effect of the steroid on human neonatal monocytes (NMs). This member of the short chain alcohol dehydrogenase catalyses the reversible inactivation of PGs into their respective 15-keto metabolites (Anggard, 1966). We show here that this enzyme could play an important role in the vitamin D3 in vitro induced differentiation of NMs through the macrophage-osteoclast lineage.