Pathologic thrombopoiesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is frequently complicated by thrombocytosis correlated with disease activity. The exact pathogenetic mechanism(s) that cause increased platelet counts in RA are still unknown. Recent investigations indicate that proinflammatory pleiotropic cytokines of RA also have megakaryocytopoietic/thrombopoietic properties. Moreover, several lineage-dominant hematopoietic cytokines can also act as acute phase responders and contribute to the inflammation. This review focuses on the current literature and our experience regarding the dual relationships of the pathologic thrombopoiesis of RA. Growth factors contributing to it, namely interleukin (IL)-6, IL-11, stem cell factor, leukemia inhibitory factor, granulocyte colony stimulating factor, thrombopoietin (TPO), and the regulation of megakaryocytopoiesis during the inflammatory cascade are reviewed. Some data indicate that thrombopoietin could contribute to the reactive thrombocytosis of RA. In the non-lineage-specific gp130 cytokine family, IL-6 appears to predominate for the induction of megakaryopoiesis. However, other cytokines and growth factors may also contribute to the pathologic megakaryocytopoiesis of RA. Those pleiotropic mediators seem to act in concert to regulate this enigmatic process. Clarification of the pathobiologic basis of thrombopoiesis in RA may improve understanding of the disease pathogenesis and management of the inflammatory thrombocytosis.     

The pathogenesis of rheumatoid arthritis: a guide to therapy

The cause of rheumatoid arthritis (RA) is unknown; however, extensive research has yielded great insight into its pathogenesis. Lymphocytes play a significant role, but a lesser role in the perpetuation of late disease. The rheumatoid synovium is composed primarily of fibroblasts and monocytes that produce inflammatory cytokines, of which interleukin-1 and tumor necrosis factor are of key importance. Potential regulatory mechanisms balancing the effects of these cytokines are inadequate to prevent joint damage and subsequent disability. These cytokines seem responsible for stimulating destructive processes in the joint via induction of prostaglandins, angiogenesis, chemokines, adhesion molecules, osteoclastogenesis, and matrix metalloproteinases. This review discusses recent research findings in the immunopathogenesis of RA with respect to potential targets for therapy.     

Mechanism of bone destruction in rheumatoid arthritis

Osteoclasts play a critical role in bone destruction in rheumatoid arthritis. Activation of osteoclastogenesis is mediated by the enhanced expression of RANKL (receptor activator of NF-kappaB ligand), accompanied by reduced expression of its inhibitor, IFN-gamma. Accumulating evidence indicates that the osteoclast-targeted therapy is effective in arthritis models, suggesting a promising new strategy for rheumatoid bone destruction.     

Mollaret’s meningitis: 65 years of history

There is an emerging interest in the role of anti-TNF-α therapy in reducing bone damage in chronic arthritis with special regard to rheumatoid arthritis. Accumulation of osteoclasts in rheumatoid synovial tissues, and their activation due to osteoclastogenic cytokines and chemokines at cartilage erosion sites suggest that they may advantageously be considered as therapeutic targets. Given that the primary role of TNF-α in osteoclastogenesis, the inhibition of TNF-α represents an important strategy for reducing bone damage in rheumatoid arthritis. In point of fact, there is evidence that treatment with anti-TNF-α agents may avoid or reduce bone damage in rheumatoid arthritis, even if further studies are required to provide a biological explanation and a link for the observation of the advantageous effects of TNF-α inhibitors on the progression of bone damage in chronic arthritis. The existence of factors involved in osteoclast activation, including IL-1, IL-6, IL-7, IL-11, IL-17, M-CSF, TGF-β, MIP-1α, MIP-1β, IP-10, MIG, and OSCAR, indicates that TNF-α is only a single player in the great molecular cauldron of osteoclastogenesis. The presence of mediators behind the TNF-α and RANK-RANKL complex that may be independent in inducing osteoclastogenesis, such as NFATc1, suggests that the anti-TNF-α therapy will not provide a complete reduction of bone damage in chronic arthritis.     

In vitro studies of megakaryocytopoiesis in thrombocytotic disorders of man

Increased numbers of bone marrow megakaryocytes and thrombocytosis are frequently observed in patients with myeloproliferative disorders (MPD). Increased marrow megakaryocytes and thrombocytosis are also noted in a variety of inflammatory and neoplastic disease leading to the phenomenon of reactive thrombocytosis (RT). The pathogenesis of this finding remains incompletely understood. Using methodology developed in our laboratory, we investigated the causative role of megakaryocyte colony-stimulating activity (Meg-CSA) in generating this phenomenon. We also examined the cloning efficiency of colony-forming units-megakaryocyte (CFU-M) and their responsiveness to an exogenous source of Meg-CSA in patients with these diseases. The results of our investigations suggest that: (1) increased production of Meg-CSA is not responsible for the megakaryocyte hyperplasia and thrombocytosis noted in these patients; (2) the intrinsic stem cell defect described in MPD appears to affect the CFU-M of these patients as well, resulting in an effective expansion of the CFU-M pool with consequent megakaryocyte hyperplasia and thrombocytosis; (3) the CFU-M of patients with MPD remain responsive to an exogenous source of Meg-CSA, suggesting that this megakaryocyte hyperplasia may not be entirely autonomous of its effects; and (4) the CFU-M pool in RT is normal both in size and responsiveness to Meg-CSA, suggesting that in these disorders, the stimulus leading to megakaryocyte hyperplasia and thrombocytosis is active at the post-CFU-M level of megakaryocyte differentiation.     

Osteoporosis en la artritis psoriásica

Psoriatic arthritis (PA) is defined as an inflammatory form of arthritis associated with psoriasis. This condition is mainly characterized by the presence of arthritis, enthesitis, typical radiographic findings, and the absence of rheumatoid factor. The increase in immune and inflammatory cells of the synovitis produces cytokines and other molecules that may influence bone remodelling, increasing the frequency of osteoporosis and fragility fractures. Osteoporosis has been better documented in rheumatoid arthritis than in PA and there is less information on the bone mass of PA patients. Osteoporosis in PA has been studied less extensively and in heterogeneous groups of patients, and the results are contradictory since not all studies have shown a higher incidence of osteoporosis. The present review summarizes the suggested etiopathogenic mechanisms and possible strategies in the clinical management and treatment of this entity and provides a review of the studies published on the topic. The main mechanisms involved in osteoporosis in PA would seem to be the activation of osteoclastogenesis by the inflammatory disease itself and corticosteroid therapy. PA is a common condition in clinical practice and it more data on osteoporosis in these patients would help to improve decision making in the management of the PA.     

Involvement of fibroblast growth factor-2 in joint destruction of rheumatoid arthritis patients.

OBJECTIVE: To investigate the effect of the synovial fluid from knee joints of rheumatoid arthritis (RA) patients with different severities of joint destruction on osteoclastogenesis and bone resorption. METHODS: Synovial fluid was harvested from the knee joints of 59 RA patients and 37 ostcoarthritis (OA) patients. RA patients with Larsen's knee grade 1-3 were classified as mild RA (n = 30) and those with grade 4 or 5 as severe RA (n = 29). Cytokine concentrations in synovial fluid were measured by ELISA. Osteoclastogenesis was measured by tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation in a co-culture of mouse osteoblastic cells and bone marrow cells, and bone resorption by 45Ca release from pre-labelled cultured neonatal mouse calvariae. RESULTS: The synovial fluid of severe RA patients significantly stimulated TRAP-positive MNC formation and 45Ca release compared to those of mild RA and OA patients. Among the bone-resorptive cytokines fibroblast growth factor-2 (FGF-2), tumour necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-6 and soluble IL-6 receptor (sIL-6R), only FGF-2 concentration in the synovial fluid was positively correlated to Larsen's grade, and severe RA patients showed significantly higher FGF-2 concentrations than mild RA patients. Osteoclastogenesis in a co-culture system which was stimulated by the synovial fluid of severe RA patients was significantly inhibited by a neutralizing antibody against FGF-2 and this inhibition was stronger than antibodies against other cytokines. CONCLUSION: The increase in endogenous FGF-2 levels in the synovial fluid of RA patients may play a role in the joint destruction by inducing osteoclastogenesis.     

Bloodstream thrombopoietin in rheumatoid arthritis with thrombocytosis

Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. Recent studies have shown that TPO may also act as an acute-phase reactant, and it has been suggested as a component of inflammatory reactions. In this study our objective was to investigate serum TPO levels in patients with rheumatoid arthritis, a complex chronic inflammatory disorder not uncommonly associated with thrombocytosis. Bloodstream TPO concentrations were assessed in 13 RA patients with platelet counts between 450 and 650 x 10(9)/l, 10 RA patients with platelet counts >650 x 10(9)/l, 15 RA patients with normal platelet counts and 12 healthy controls. RA patients with normal platelet counts had TPO levels comparable with healthy controls. TPO concentrations in patients with mild thrombocytosis were significantly elevated, whereas patients with markedly increased thrombocyte counts had prominently decreased TPO levels. These results indicate that TPO seems to be associated with reactive thrombocytosis in RA patients with active disease. In patients with extremely increased thrombocytosis serum TPO levels might be regulated by increased platelet mass via receptor-mediated uptake and metabolism.     

Anomalous megakaryocytopoiesis in mice with mutations in the c-Myb gene

Mpl(-/-) mice bearing the Plt3 or Plt4 mutations in the c-Myb gene exhibit thrombopoietin (TPO)-independent supraphysiological platelet production accompanied by excessive megakaryocytopoiesis and defective erythroid and lymphoid cell production. To better define the cellular basis for the thrombocytosis in these mice, we analyzed the production and characteristics of megakaryocytes and their progenitors. Consistent with thrombocytosis arising from hyperactive production, the high platelet counts in mice carrying the c-Myb(Plt4) allele were not accompanied by any significant alteration in platelet half-life. Megakaryocytes in c-Myb mutant mice displayed reduced modal DNA ploidy and, among the excessive numbers of megakaryocyte progenitor cells, more mature precursors were particularly evident. Megakaryocyte progenitor cells carrying the Plt3 or Plt4 c-Myb mutations, but not granulocyte-macrophage progenitors, exhibited 200-fold enhanced responsiveness to granulocyte-macrophage colony-stimulating factor (GM-CSF), suggesting that altered responses to cytokines may contribute to expanded megakaryocytopoiesis. Mutant preprogenitor (blast colony-forming) cells appeared to have little capacity to form megakaryocyte progenitor cells. In contrast, the spleens of irradiated mice 12 days after transplantation with mutant bone marrow contained abundant megakaryocyte progenitor cells, suggesting that altered c-Myb activity skews differentiation commitment in spleen colony-forming units (CFU-S) in favor of excess megakaryocytopoiesis.     

Hematopoietic stromal cells and megakaryocyte development

The hematopoietic microenvironment, and in particular the hematopoietic stromal cell element, are intimately involved in megakaryocyte development. The process of megakaryocytopoiesis occurs within a complex bone marrow microenvironment where adhesive interactions, chemokines, as well as cytokines play a pivotal role. Here we review the effect of stromal cells and cytokines on megakaryocytopoiesis with the aim of exploring new therapeutic strategies for platelet recovery after hematopoietic stem cell transplantation (HSCT).     

Estrogen deficiency accelerates murine autoimmune arthritis associated with receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenesis

The aims of this study were to evaluate the in vivo effects of estrogen deficiency in MRL/lpr mice as a model for rheumatoid arthritis and to analyze the possible relationship between immune dysregulation and receptor activator of nuclear factor-kappaB ligand (RANKL)-mediated osteoclastogenesis. Experimental studies were performed in ovariectomized (Ovx)-MRL/lpr, Ovx-MRL+/+, sham-operated-MRL/lpr, and sham-operated-MRL+/+ mice. Severe autoimmune arthritis developed in younger Ovx-MRL/lpr mice until 24 wk of age, whereas these lesions were entirely recovered by pharmacological levels of estrogen administration. A significant elevation in serum rheumatoid factor, anti-double-stranded DNA, and anti-type II collagen was found in Ovx-MRL/lpr mice and recovered in mice that underwent estrogen administration. A high proportion of CD4(+) T cells bearing RANKL was found, and an enhanced expression of RANKL mRNA and an impaired osteoprotegerin mRNA was detected in the synovium. An increase in both osteoclast formation and bone resorption pits was found. These results indicate that estrogen deficiency may play a crucial role in acceleration of autoimmune arthritis associated with RANKL-mediated osteoclastogenesis in a murine model for rheumatoid arthritis.     

Treatment of bone metastases

Enhanced osteoclastogenesis and osteoclastic activity through receptor activator of nuclear factor-kappa B (RANK) ligand - RANK system together with cytokines released from bone matrix during bone resorption play important roles in the development of bone metastases. Bisphosphonates with potent inhibitory activity on bone resorption have been shown to reduce the number of skeletal events and have some analgesic effect in patients with bone metastases from breast cancer or multiple myeloma. However, bisphosphonates do not affect survival of these patients and better drugs for prevention and treatment of bone metastases are necessary.     

Circulating Thrombopoietin in Clonal Versus Reactive Thrombocytosis

Introduction: The aim of this study is to assess circulating thrombopoietin concentrations in patients with both clonal and reactive thrombocytosis (RT), which are two distinct categories of extreme platelet production circumstances. Investigation of the thrombopoietin levels in clonal versus reactive thrombocytosis may help us to understand the interactions of this key regulatory cytokine and the conditions in which abnormally increased platelet formation exist.

Materials and methods: Thrombopoietin levels were measured in patients with platelet counts greater than 500 × 10³ μl^?1 The study population consisted of 21 patients with RT (13 with iron deficiency anemia, and 8 with rheumatoid arthritis), 24 patients with clonal thrombocytosis (six with essential thrombocytosis, three with myelofibrosis, eight with chronic myelogenous leukemia, and seven with polycythemia vera (PV)) and 16 healthy subjects were used as controls.

Results: The median plasma thrombopoietin concentration was 100.5 pg ml^?1 in patients with RT, 467pg ml^?1 in patients with clonal thrombocytosis and 62.65pgml^?1 in the control group. The thrombopoietin concentration was found to be higher in the patients with primary thrombocytosis when compared to the control group (p = 0.001), as well as in patients with RT (p = 0.002). However, there was no statistically significant difference between the patients with RT and the control group (p = 0.14). There was no correlation between thrombopoietin levels and the platelet counts in patients with clonal thrombocytosis, including essential thrombo- cythemia (ET).

Conclusion: Increased levels of thrombopoietin were found in patients with clonal thrombocytosis versus patients with RT and control subjects as well. Defective clearance of thrombopoietin by megakaryocytes and platelets due to a reduced number of thrombopoietin receptors may be the causative mechanism behind this. These results indicate that plasma thrombopoietin levels may be helpful in distinguishing between clonal and reactive thrombocytosis.     

Hematopoietic stromal cells and megakaryocyte development

Abstract

The hematopoietic microenvironment, and in particular the hematopoietic stromal cell element, are intimately involved in megakaryocyte development. The process of megakaryocytopoiesis occurs within a complex bone marrow microenvironment where adhesive interactions, chemokines, as well as cytokines play a pivotal role. Here we review the effect of stromal cells and cytokines on megakaryocytopoiesis with the aim of exploring new therapeutic strategies for platelet recovery after hematopoietic stem cell transplantation (HSCT).     

miRNA-146a in rheumatoid arthritis: a new therapeutic strategy

Evaluation of: Nakasa T, Shibuya H, Nagata Y, Niimoto T, Ochi M: The inhibitory effect of microRNA-146a expression on bone destruction in collagen-induced arthritis. Arthritis Rheum. 63(6), 1582-1590 (2011). miRNA-146a (miR-146a) has been shown to play an important role in the negative regulation of inflammatory innate immune responses, and is differentially expressed in a number of human diseases including rheumatoid arthritis. However, evidence for the potential therapeutic use of miR-146a in human disease has been lacking. The current paper demonstrates the potential therapeutic application of miR-146a for rheumatoid arthritis by demonstrating the inhibitory effect of miR-146a on osteoclastogenesis in vitro. Moreover, using a collagen-induced arthritis mouse model, they were able to demonstrate that intravenous administration of double-stranded miR-146a resulted in the suppression of cartilage and bone destruction, despite relatively unaffected immune cell infiltration of the synovium and inflammatory cytokine expression.     

Cytokine production by primary bone marrow megakaryocytes

Primary human bone marrow megakaryocytes were studied for their ability to express and release cytokines potentially relevant to their proliferation and/or differentiation. The purity of the bone marrow megakaryocytes was assessed by morphologic and immunocytochemical criteria. Unstimulated marrow megakaryocytes constitutively expressed genes for interleukin-1 beta (IL-1 beta), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), by the polymerase chain reaction (PCR) and Northern blot analysis. At the protein level, megakaryocytes secreted significant amounts of IL-1 beta (53.6 +/- 3.6 pg/mL), IL-6 (57.6 +/- 15.6 pg/mL), and GM-CSF (24 +/- 4 pg/mL) but not TNF-alpha. Exposure of human marrow megakaryocytes to IL-1 beta increased the levels of IL-6 (87.3 +/- 2.3 pg/mL) detected in the culture supernatants. Transforming growth factor- beta was also able to stimulate IL-6, IL-1 beta, and GM-CSF secretion, but was less potent than stimulation with phorbol-12-myristate-13- acetate (PMA). The secreted cytokines acted additively to maintain and increase the number of colony-forming unit-megakaryocytes colonies (approximately 35%). These studies demonstrate the production of multiple cytokines by isolated human bone marrow megakaryocytes constitutively or stimulated in vitro. The capacity of human megakaryocytes to synthesize several cytokines known to modulate hematopoietic cells supports the concept that there may be an autocrine mechanism operative in the regulation of megakaryocytopoiesis.     

The frequency and significance of megakaryocytic emperipolesis in myeloproliferative and reactive states

Summary Sixty-three bone marrow (BM) biopsy paraffin sections from patients with platelet counts of 1000×10⁹/1 or greater were examined to determine the incidence of megakaryocytic emperipolesis for the various myeloproliferative disorders (MPDs) and for reactive thrombocytosis. Of those cases classified as specific MPDs, 77% of primary thrombocythemia (PT) specimens, 100% of the polycythemia vera (PV) specimens, a single idiopathic myelofibrosis (IMF) specimen, and 17% of the chronic granulocytic leukemia (CGL) specimens demonstrated emperipolesis within megakaryocytes. Two of three cases grouped as MPDs but not further classified also demonstrated emperipolesis. Of the cases of reactive thrombocytosis (RT), 75% showed the presence of emperipolesis. Our results indicate that, with the exception of CGL, emperipolesis can be found in the BM megakaryocytes of the great majority of patients who have extreme thrombocytosis. The underlying cause, whether myeloproliferative or reactive, does not apparently influence the incidence of the phenomenon.     

Involvement of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis

OBJECTIVE: To clarify the mechanism by which osteoclasts are formed in culture of rheumatoid synoviocytes by exploring the involvement of receptor activator of nuclear factor kappaB ligand (RANKL)/osteoclast differentiation factor (ODF). METHODS: Osteoclast formation was evaluated in cocultures of rheumatoid synovial fibroblasts and peripheral blood mononuclear cells (PBMC) in the presence of macrophage colony stimulating factor and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) utilizing separating membrane filters. RANKL/ODF expression was examined by Northern blotting in synovial tissues from 5 rheumatoid arthritis (RA) patients and tissues from patients with giant cell tumor (GCT), osteosarcoma (OS), and osteoarthritis (OA). RANKL/ODF expression and the ability of synovial fibroblasts to support osteoclastogenesis were investigated in coculture with PBMC in the presence or absence of 1,25(OH)2D3, and soluble RANKL/ODF and osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) were measured by enzyme-linked immunosorbent assay. The effects of OPG/OCIF on the osteoclastogenesis in the primary culture of rheumatoid synoviocytes and the coculture system were determined. RESULTS: Synovial fibroblasts did not induce osteoclastogenesis when separately cocultured with PBMC. Northern blotting revealed that RANKL/ODF was highly expressed in all tissues from RA and GCT patients, but not from OA or OS patients. Cultured rheumatoid synovial fibroblasts efficiently induced osteoclastogenesis in the presence of 1,25(OH)2D3, which was accompanied by up-regulated expression of RANKL/ODF and decreased production of OPG/OCIF. Osteoclastogenesis from synoviocytes was dose-dependently inhibited by OPG/OCIF. CONCLUSION: RANKL/ODF expressed on synovial fibroblasts is involved in rheumatoid bone destruction by inducing osteoclastogenesis and would therefore be a good therapeutic target.