Effects Of Zoledronic Acid On Lymphocyte Subpopulations In Patients With Primary Osteoporosis In Vivo

IntroductionOsteoporosis is the most common bone disease and represents a major public health problem.Osteoporosis is characterized by a systemic impairment of bone mass and of the microarchitecture that results in fragility fractures.It affects about 20%of women and 7%of men aged over 50 years,with half of them suffering an osteoporotic fracture,particularly hip,wrist and vertebral fractures.From the point of view of cellular pathophysiology,osteoporosis results from a preponderance of activity of osteoclasts over that of osteoblasts.Traditionally,osteoporosis has been regarded as an endocrine(in particular,estrogen deficiency-mediated)disease;nevertheless,it is also well established that chronic inflammatory diseases(e.g.rheumatoid arthritis,ankylosing spodylitis,Cron's disease,ulcerative colitis,chronic obstructive pulmonary disease and certain viral infections)are associated with osteoporotic fractures.Clinical and experimental findings evidenced a close connection between osteoporosis and immune deficiency syndrome,and a common inflammatory background has been finally discovered as pathogenetic factor even in conditions of major osteoporotic risk,such as old age and estrogen deficiency.The main pathogenetic mechanism leading to bone tissue alteration seems to be inflammation.From this point of view osteoporosis could be therefore regarded as an immune mediated disease in which immune activation,through the induction of cytokine production and inflammation,leads to a remodeling of osteoclast cells and osteoblast cells and dysregulation of bone turnover with consequent increased bone resporption and osteoporosis.Bone and immune system are functionally intergrated through complex homeostatic networks and in all respects,osteoporosis could be considered a chronic immune mediated inflammatory disease which shares clinical and biological features with many other immune mediated diseases.Over the years,our knowledge about the cross talk between the immune system and osseous tissue has significantly increased;in 2000,the term osteoimmunology was coined.Since then,osteoimmunology has become a prosperous and promising research field.Thus osteoimmunology appears definitely as an interdisciplinary research and clinical field which also allowed new pathogenetic and clinical interpretations of well-known and common diseases,such as osteoporosis.Its fields of interest are constantly expanding,thus enriching with an increasing number of translational implications,even in clinical practice and in various branches of medicine.Here the most important concepts in osteoimmunology are addressed in the context of physiopathological states bridging these two organ systems,including osteoporosis,ageing,menopause,inflammatory arthritis,cancer,dysmetabolism and neurological disorders.Many molecules impact on the balance of bone resorption by osteoclasts and bone formation by osteoblasts.Osteoblasts produce positive and negative regulation of osteoclastogenesis such as receptor activator of nuclear factor(NF)-KB ligand(RANKL)and the natural decoy receptor for RANKL,osteoprotegerin(OPG),respectively.Osteoclast differentiation is initiated by binding of RANKL,a TNF family cytokine,to te receptor activator of NF-Kb(RANK).Osteoblasts are a major source of RANKL,but the cytokine is also expressed by osteocytes,fibroblasts or cells of the immune system,including antigen-stimulated T cells and mature dendritic cells.Immune activation may directly induce osteoclastogenesis and bone resorption through RANKL.The cells of the immune system,mainly activated T and B lymphocytes and dendritic cells,express RANKL.Morever,RANKL,the principal osteoclastogenic mediator,stimulates bone resorption through the NFATc1,which is also a crucial factor in the immune system regulation.T lymphocytes resident in the bone marrow are te key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone,in physiological and pathological conditions.During inflammatory diseases or in conditions characterized by low-grade systemic inflammation,such as menopause and aging.Osteoclast bone resorption is driven by inflammatory cytokines produced by activated T lymphocytes.However,bone marrow T cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells.Two mechanisms are involved:the binding of T cell costimulaory molecules to their counter receptors on.bone cells and their precursors,and the release of cytokines and Wnt ligands that activate Wnt signaling in osteoblastic cell lineage.The final effect of T lymphocytes on bone depends on their activation state and their specific phenotype.Not only the immune system regulates bone remodeling,but also the bone is able to influence the immune system,actively interacting with immune cells.The same bone cells would then be able to influence or even also perform many immune functions,such as cytokine production and antigen presentation.In this sense,the bone would be regarded as a sort of expanded immune system.Amino-bisphosphonates are now established therapies for osteoporosis and Paget disease,and they are widely used for the prevention and treatment of skeletal-related events in cancer.Zoledronic acid is a bisphosphoate that contains an imidazole ring in a side chain,which,with once-yearly intravenous infusion,has been shown to cause an increase in bone mineral density that is mediated by a potent inhibitory effect on bone resorption.Zoledronic acid has already been approved in a number of countries for postmenopausal osteoporosis,glucocorticoid-induced osteoporosis,and male osteoporosis.The use of intravenous zoledronic acid is occasionally associated with the appearance within 24-36 h of fever and musculoskeletal pain;this is referred as the acute phase response and it is associated with a transitory fall in circulating lymphocyte number.It is known that the acute phase response is linked to the activation of ??T cells and the release of pyrogenic cytokines.Studies have revealed that amino-bisphosphonates act via accumulation in adjacent monocytes of intracellular upstream metabolites,including isopentenyl pyrophosphate,after amino-bisphosphonates mediated inhibition of farnesyl pyrophosphate synthase.It has been observed that both IV or oral amino-bisphosphonates treatment is associated with a decrease in circulating ??T cells for at least 1 year.Recently,Kalyan et al.confirmed a long-term loss of ??T cells in osteoporotic patients on oral amino-bisphosphonates and even more striking decline in patients administered IV amino-bisphosphonates.It has been observed that IV zoledronic acid treatment is associated with a decrease not only in circulating ??T cells,but also of total WBC,lymphocytes,CD4+T cell,and eosinophils.The clinical implication of a persistent decrease in circulating ??T cells might be of great interest.The ??T cells represent only 1-10%of CD3+ T cells in the human peripheral circulation,although their number is more abundant in epithelial tissues.It may also ensue as a result of the hematopoietic stem cell.Kalian et al.documented a significant decrease in ??T cells in patients who had experienced zoledronic acid-associated osteonecrosis of the jaw(BAONJ),and they hypothesized that BAONJ is a consequence of the drug-induced immune long-term effect.Accumulating evidence over the past decade suggest a model of extensive interation between immune system and bone.Zoledronic acid is a probably immunotherapy for primary osteoporosis.ObjectiveTo investigate the effects of zoledronic acid on lymphocyte and immune system in patients with primary osteoporosis,through observing the blood cells analysis,the level of lymphocyte subpopulations,before and on day 1,2 and 3 after 1st,2nd administration of intravenous zoledronic acid and lymphocyte subpopulations,bone turnover markers and bone mineral destiny 1 year after a single administration of zoledronic acid.Material and Methods1.Patients:We consecutively enrolled 30 patients affected by primary osteoporosis followed at the pain department of Jinan Central Hospital.Inclusion critera included:patients who had been diagnosed with primary osteoporosis,no administration of amino-bisphosphonates before,no serious complications including the liver disease,serum calcium and phosphorus in the normal level;creatinine clearance>35ml/min,exclusion critera included history of autoimmune or hematological diseases or cancer or acute infections;a body temperature>38.5?.The patients received 2 annual intravenous infusions of zoledronic acid 5mg.All subjects received daily oral supplements of 600mg calcium and 0.5?g calcitriol.2.Parameters involved:2.1 Before and lyear after the administration of intravenous zoledronic acid.Bone mineral density of the lumbar spine was monitored using QCT,the bone turnover markers:OC,PTH,?-CTX and 25(OH)D were measured by Elecsys.The blood cells analysis and the blood electrolyte were tested.The level of lymphocyte subpopulations was measured by flow cytometry.2.2 Before and Id,2d,3d after 1st and 2nd administration of intravenous zoledronic acid.The blood cells analysis were performed by an automated hematology analyzer and the blood electrolyte were tested by an automatic biochemical analyzer.The level of lymphocyte subpopulations was measured by flow cytometry.ResultsA total of 30 subjects were screened in our study.All the patients(male:9,female:21)completed the 1st administration of intravenous zoledronic acid.The average age was(73.28±6.44)years old.Of these,26 subjects(male:7,female:19)completed the 2nd administration of intravenous zoledronic acid.The average age was(75.21±5.01)years old.1.The BMD T score of lumbar spine increased from(-3.78±0.76)to(-2.83±0.65)after lyear of zoledronic acid treatment.Serum ?-CTx significantly decreased,whereas the other bone turnover markers showed no significant changes.The peripheral cell count,serum ion level and lymphocyte subpopulation showed no significant change.2.1 Significant increase of total WBC,NEU and NEU%was observed on day 1 after the 1st infusion of zoledronic acid in the osteoporosis patients than the baseline,while the level of LYM,LYM%,EOS,EOS%significantly decreased(P<0.01).The level of serum sodium,calcium,phosphorus was lower than the baseline after the 1st infusion of zoledronic acid.No significant differences in the level of serum kalium and chlorine.Patients showed a decreased of the count of CD3+ cells,CD3+CD4+ cells,CD3+CD4+/CD3+CD8+ value and CD 16+CD56+ cells,while the count of CD3+CD8+cells and CD 19+ cells showed no difference.2.2 Significant increase of NEU%was observed on day 2 after the 1st infusion,while the level of other cells showed no significant difference.The level of serum sodium,kalium,calcium,phosphorus was still lower than the baseline.No significant differences in the level of serum chlorine.The level of CD3+ cells,CD3+CD4+ cells,CD3+CD4+/CD3+CD8+ value and CD16+CD56+ cells still significantly decreased,while the count of CD3+CD8+ cells and CD19+ cells showed no difference.2.3 Significant increase of total WBC and LYM count was observed on day 3 after the 1st infusion,while the level of other cells showed no significant difference.The level of serum calcium and phosphorus was still lower than the baseline.No significant differences in the level of serum sodium and kalium.The level of CD16+CD56+ cells still significantly decreased,while the other cells showed no difference.3.1 Significant increase of NEU%was observed on day 1 after the 2nd infusion of zoledronic acid,while the other cell counts showed no difference.The peripheral cell count,serum ion level and lymphocyte subpopulation showed no significant change.3.2 Significant increase of the total WBC?NEU?NEU%was observed on day 2 after the 2nd infusion of zoledronic acid than the baseline,while the LYM%?EOS%significantly decreased(P<0.01).3.3 The peripheral cell count,serum ion level and lymphocyte subpopulation was observed on day 3 after the 2nd infusion of zoledronic acid showed no significant change than the baseline.Conclusion1.Osteoporosis patients after the first infusion with zoledronic acid in a 3-day period showed a significant increase of NEU and NEU%,a significant decrease of LYM,LYM%,EOS,EOS%and CD3+ cell.CD3+CD4+ cell.CD3+CD4+/CD3+CD8+ ratio?CD16+CD56+ cell(P<0.01).These results suggest that zoledronic acid may have immunomodulatory effects through affecting lymphocyte subpopulation.2.The BMD T score of lumbar spine increased,serum bone resorption marker decreased after lyear of zoledronic acid treatment,whereas the peripheral cell count,serum ion level and lymphocyte subpopulation showed no significant change than the baseline.3.ratients with primary osteoporosis after the 2nd infusion of zoledronic acid in a 3-day period showed a significant increase of NEU and NEU%,a significant decrease of LYM,LYM%,while CD3+CD4+ cell only decreased on day 2 after the treatment(P<0.01).These results suggest that zoledronic acid induced acute phase response may be associated with the transitory change of lymphocyte subpopulation.These results suggest that immunoeffect of zoledronic acid is under control,without inducing the imbalance of immune system.Our study opens a new frontier for the understanding of the immunoeffects of zoledronic acid.