Chronic lymphocytic leukemia cells impair osteoblastogenesis and promote osteoclastogenesis: role of TNFα, IL-6 and IL-11 cytokines

Abstract

Bone skeletal alterations are no longer considered a rare event in chronic lymphocytic leukemia (CLL), especially at more advanced stages of the disease. This study is aimed at elucidating the mechanisms underlying this phenomenon. Bone marrow stromal cells, induced to differentiate toward osteoblasts in osteogenic medium, appeared unable to complete their maturation upon co-culture with CLL cells, CLL-cell-derived conditioned media (CLL-cm) or CLL-sera (CLL-sr). Inhibition of osteoblast differentiation was documented by decreased levels of RUNX2 and osteocalcin mRNA expression, by increased osteopontin and DKK-1 mRNA levels, and by a marked reduction of mineralized matrix deposition. The addition of neutralizing TNFα, IL-11 or anti-IL-6R monoclonal antibodies to these cocultures resulted in restoration of bone mineralization, indicating the involvement of these cytokines. These findings were further supported by silencing TNFα, IL-11 and IL-6 in leukemic cells. We also demonstrated that the addition of CLL-cm to monocytes, previously stimulated with MCSF and RANKL, significantly amplified the formation of large, mature osteoclasts as well as their bone resorption activity. Moreover, enhanced osteoclastogenesis, induced by CLL-cm, was significantly reduced by treating cultures with the anti-TNFα monoclonal antibody infliximab. An analogous effect was observed with the use of the BTK inhibitor, ibrutinib. Interestingly, CLL cells co-cultured with mature osteoclasts were protected from apoptosis and upregulated Ki-67. These experimental results parallel the direct correlation between amounts of TNFα in CLL-sr and the degree of compact bone erosion that we previously described, further strengthening the indication of a reciprocal influence between leukemic cell expansion and bone structure derangement.

A High Percentage of CD16+ Monocytes Correlates with the Extent of Bone Erosion in Chronic Lymphocytic Leukemia Patients: The Impact of Leukemic B Cells in Monocyte Differentiation and Osteoclast Maturation

“…The low-affinity receptor FcγRIIIa, or CD16, may facilitate actin cytoskeleton re-organization and may contribute in regulating multiple-cell fusion together with other RANK co-receptors [ 29 , 30 ]. Since we previously determined enhanced osteoclast formation from monocytes cultured with media derived from CLL B cell cultures (CLL-cm) [ 4 ], we evaluated here whether healthy monocytes, cultured with CLL-cm, up-regulated the expression of CD16, RANK and RANKL. As shown in Figure 4 A,B, the addition of CLL-cm significantly increased CD16 expression in monocytes derived from healthy donors.…”

“…We have shown that monocytes from normal donors, cultured in the presence of a CLL-conditioned medium, upregulated Indoleamin-2,3 dioxygenase (IDO) and that IDO expression is significantly higher in monocytes from CLL patients than in monocytes from normal controls [ 1 ]. In addition, we recently reported that leukemic CLL cells also affect the differentiation of the two major cellular components of the bone tissue, osteoblasts and osteoclasts, leading to deregulation of bone remodeling [ 4 ].…”

“…The expression of CD16, RANK or RANKL molecules was evaluated on purified monocytes obtained from healthy donors, before and after treatment with CLL-conditioned media (CLL-cm) derived from various patients. Monocytes were purified from PBMCS of healthy donors by the use of anti-CD14 microbeads (Miltenyi Biotech GMbH Friederich-Ebert, Germany), as previously described [ 4 ], and were cultured in 24-well plates with basal medium only (RPMI 1640 +3% FCS) (Lonza, Walkersville, MD, USA) or with the addition of CLL-conditioned media (CLL-cm: 300 µL) for 48 h or for different times as indicated. CLL-cm were derived from B CLL cell culture as previously described [ 4 ].…”

“…Monocytes were purified from PBMCS of healthy donors by the use of anti-CD14 microbeads (Miltenyi Biotech GMbH Friederich-Ebert, Germany), as previously described [ 4 ], and were cultured in 24-well plates with basal medium only (RPMI 1640 +3% FCS) (Lonza, Walkersville, MD, USA) or with the addition of CLL-conditioned media (CLL-cm: 300 µL) for 48 h or for different times as indicated. CLL-cm were derived from B CLL cell culture as previously described [ 4 ]. Cells were then detached from each well by gentle scraping, double-stained with the anti- human CD14-FITC and CD16-PE moAbs (Immunotools) and incubated at 4 °C for 30 min in the dark.…”

“…Interactions between CLL cells and accessory cells of the microenvironment, as well as the dysregulation of the immune system control, contribute to increasing leukemic cells survival [ 1 , 2 ], further causing phenotypical and functional modifications in the interacting cell types [ 3 ]. We recently demonstrated that CLL cells inhibited osteoblast differentiation and stimulated osteoclast formation; osteoclasts, in turn, enhanced CLL cell viability and proliferation [ 4 ], thus, creating a reciprocal self-sustaining loop. The observation that patients with an advanced disease stage (Binet C versus A) [ 5 ] have higher levels of bone erosion at the long bone shaft further suggests that the progressive expansion of leukemic B cells affects physiological remodeling of the bone tissue.…”