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Endocrinology Vol. 144, No. 5 1761-1769
Copyright © 2003 by The Endocrine Society

CD40 Ligand Blocks Apoptosis Induced by Tumor Necrosis Factor {alpha}, Glucocorticoids, and Etoposide in Osteoblasts and the Osteocyte-Like Cell Line Murine Long Bone Osteocyte-Y4

Seema S. Ahuja, Shujie Zhao, Teresita Bellido, Lilian I. Plotkin, Fabio Jimenez and Lynda F. Bonewald

Department of Medicine (S.S.A., S.Z., F.J., L.F.B.) and Departments of Biochemistry and Cellular and Structural Biology (L.F.B.), University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284; South Texas Veterans Health Care System (S.S.A.), Audie L. Murphy Division, San Antonio, Texas 78284; Division of Endocrinology and Metabolism and Center for Osteoporosis and Metabolic Bone Diseases (T.B., L.I.P.), University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Address all correspondence and requests for reprints to: Lynda F. Bonewald, Ph.D., Department of Oral Biology, University of Missouri at Kansas City School of Dentistry, 650 East 25th Street, Kansas City, Missouri 64108-2784. E-mail: bonewaldl{at}umkc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During characterization of the osteocyte-like murine long bone osteocyte-Y4 (MLO-Y4) cell line, comparison was made with antigen-presenting cells of the immune system known as dendritic cells. It was observed that the MLO-Y4 osteocyte-like cells express CD40 antigen and MHC class I antigen, but they are negative for a series of other dendritic cells markers (DEC-205, CD11b, CD11c, CD86, and MHC class II) and immune cell markers [CD45, CD3, CD4, B220, Gr-1, and CD40 ligand (CD40L)]. RT-PCR results showed expression of CD40 mRNA and lack of CD40L mRNA expression. Like MLO-Y4 osteocyte cells, both primary osteoblasts and the osteoblast-like cell lines MC3T3, OCT-1, and 2T3 were shown to express CD40 antigen by fluorescence-activated cell sorting. Because CD40L has been shown to function as an antiapoptotic factor in dendritic cells, it was reasoned that this molecule may have a similar function in bone cells. In three different assays for apoptosis, including trypan blue exclusion, changes in nuclear morphology, and fluorescence-activated cell sorting staining for annexin V/propidium iodide, CD40L significantly inhibited apoptosis of MLO-Y4 cells induced by dexamethasone, TNF{alpha}, or etoposide. CD40L also inhibited dexamethasone and TNF{alpha}-induced apoptosis in the osteoblast cell lines, OCT1 and MC3T3-E1. These data support the hypothesis that CD40L preserves viability of osteoblasts and osteocytes against a wide variety of apoptotic factors independent of signaling or transcriptional mechanisms. Because osteocyte cell death appears to result in bone loss, these studies have important implications for the treatment of bone loss due to glucocorticoid excess and/or to osteoporosis in general.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE COMMITMENT TO undergo cell death can be influenced by a number of extrinsic and intrinsic cellular events. In multicellular organisms, all cells are programmed to commit suicide if survival signals are not received from their environment. These survival signals can be provided by the neighboring cells, extracellular matrix, or by growth factors (1, 2, 3, 4, 5, 6). Factors that initiate apoptosis in one cell type may block apoptosis in another cell type. For example, estrogen prevents apoptosis of osteoblastic cells but induces apoptosis of osteoclasts (7, 8).

The most common form of osteoporosis is postmenopausal, due to a lack of estrogen. Estrogen has been shown to be a viability factor or antiapoptotic factor for osteocytes in both humans and rodents (9, 10). Cytokines such as TNF{alpha} and IL-1 have been reported to increase with estrogen deficiency (11, 12). Delivery of both the soluble TNF receptor and an IL-1 receptor antagonist completely blocked bone loss due to ovariectomy in mice (13).

Apoptosis may also play an important role in the third most common cause of osteoporosis, glucocorticoid-induced osteoporosis (14). This kind of bone loss usually affects the cortical and cancellous bone of the axial skeleton. Mice administered glucocorticoids show higher numbers of apoptotic/dead osteoblasts that appear to be responsible for the decreased bone formation (15). In addition, these studies demonstrated an increase in apoptotic osteocytes that may contribute to bone fragility independent of changes in bone mass (15, 16). An increase in osteoblast/osteocyte apoptosis has also been demonstrated in patients with glucocorticoid-induced osteoporosis (17).

Dendritic cells are the most potent antigen-presenting cells known, and they initiate the adaptive immune response by interacting with naive T cells (18). Various members of the TNF receptor ligand family that serve as cell-specific survival factors or antiapoptotic factors for dendritic cells have been identified (19, 20). These include TRANCE (TNF-related activation associated cytokine), TNF{alpha}, and CD40 ligand (CD40L; Refs. 20, 21, 22). TRANCE, also known as receptor activator of nuclear factor-{kappa}B (NF-{kappa}B) ligand (RANKL), was originally described as a dendritic cell-specific survival factor expressed by T lymphocytes, and it was also described later as an osteoclast differentiation factor (23, 24). TNF{alpha} is an activator of NF-{kappa}B that acts as an antiapoptotic factor for dendritic cells and for osteoclasts but, in contrast, acts as an apoptotic factor for osteoblasts (15). CD40 and CD40L belong to a large family of receptor/ligand pairs known as the TNF superfamily that includes not only TNF receptors and TNF but also Fas/Fas ligand and CD30/CD30 ligand (6, 25). CD40L plays an important role in B cell costimulation, by inducing apoptosis of low-affinity antigen reactive cells while inducing proliferation of high-affinity antigen reactive B lymphocytes (26, 27, 28, 29).

CD40 is a 50-kDa glycoprotein expressed not only on the surface of B cells and dendritic cells but also on normal epithelium and some epithelial carcinomas (30). CD40L expressed on activated T lymphocytes, human dendritic cells, and human vascular endothelial cells may exist as a trimeric structure that induces oligomerization of its receptor upon binding. The receptor then signals by binding to members of TNF receptor-associated proteins (TRAF), such as TRAF 2 and TRAF 6, that appear to activate NF-{kappa}B transcription factor.

The CD40-CD40L signaling system clearly plays an important role in immune cell death and function, however little is known concerning the role of this signaling system in bone. In this study, we used an osteocyte-like cell line, murine long bone osteocyte-Y4 (MLO-Y4), that has characteristics of primary osteocytes (31). MLO-Y4 cells have numerous dendritic processes, the morphological feature of osteocytes in bones. They are similar to osteocytes in phenotype such as low or no expression of collagen type I and alkaline phosphatase and high expression of the bone-specific protein, osteocalcin. Interestingly, this cell line does not express an antigen specifically found on early osteoblast progenitors known as osteoblast-specific factor 2 or more recently as periostin (32, 33). These cells also express large amounts of the osteocyte-specific antigen, E11 (34), an antigen shown to localize to only osteocytes in vivo as described by Schulze et al. (35).

Until this report, the expression of CD40, the receptor for CD40L, has been shown to be limited to B cells, follicular dendritic cells, epithelial cells, hematopoietic progenitor cells, and some carcinomas. In this report, we demonstrate the following novel findings: first, that MLO-Y4 osteocyte-like cells, primary osteoblasts, and osteoblast-like cell lines express abundant CD40; and second, that addition of CD40L prevents glucocorticoid or TNF{alpha}-induced apoptosis in these bone cells. These data have important implications in bone biology and prevention of steroid-induced osteoporosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Monoclonal antibodies (mAb) and reagents
Fluorescent-labeled mAb against CD40 (clone HM40–3, 3/23), MHC class II (I-Ab haplotype, clone 25–9-3), MHC class I (H-2Kb, clone KH95), CD40L (clone MR1), CD11a (clone 2D7), CD11b (clone M1/70), CD86 (clone GL1), CD54 (ICAM-1 marker, clone 3E2), CD3{epsilon} (T cell marker, clone 145–2C11), and B220 (B cell marker, clone RA3–6B2), and the respective isotype control (hamster IgG, rat IgG2a) were obtained from PharMingen (San Diego, CA). Reagents for apoptosis staining included propidium iodide (PI; Sigma, St. Louis, MO) and annexin V fluorescein isothiocyanate (FITC; PharMingen). Anti-DEC-205 mAb (clone NLDC-145) was obtained from Caltag Laboratories, Inc. (Burlingame, CA). Soluble CD40L (trimeric form) was provided by Immunex Corp. (Seattle, WA). Tissue culture media DMEM and fetal bovine serum (FBS) was obtained from Life Technologies, Inc. (Grand Island, NY) and calf serum was from Hyclone Laboratories, Inc. (Logan, UT). Recombinant growth factors, GM-CSF, IL-4, and TNF{alpha} were obtained from R&D Systems (Minneapolis, MN). All other reagents were obtained from Sigma, unless otherwise stated.

Cell lines and culture conditions
The establishment of the MLO-Y4 cell line as an osteocyte cell line has been previously described (31). The osteoblastic cell line, OCT-1, which was established from osteocalcin promoter-driven T antigen transgenic mouse calvaria was a gift of Dr. Di Chen [University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX; Ref. 36 ]. Primary osteoblastic cells were isolated from neonatal mouse calvaria by sequential collagenase digestion according to the previously described method of Takahashi et al. (37). All animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals and with approval of the UTHSCSA Animal Care Committee. The osteoblastic cell line, MC3T3-E1, established from normal mouse calvaria, was a gift of Dr. Hiroaki Kodama (Ohu University, Fukushima, Japan; Ref. 38). Dendritic cells were derived from bone marrow as previously described (39, 40, 41). Briefly, the femurs and tibias were flushed with 3–5 ml of 1% BSA in PBS. Particulate matter was filtered, and red blood cells were lysed using red blood cell lysing buffer (Sigma). Bone marrow cells were differentiated into dendritic cells by culturing in RPMI (Life Technologies, Inc.) supplemented with 10% fetal calf serum (FCS), gentamicin (10 µg/ml), recombinant murine cytokines GM-CSF (50 ng/ml), and IL-4 (1 ng/ml) for 7–10 d. On d 3 and d 5, the nonadherent cells (contaminating granulocytes and lymphocytes) were removed and replaced with fresh medium and growth factors. On d 7, the adherent cells were scraped and plated in either 24- or 96-well plates. In several experiments, bone marrow-derived dendritic cells were stained and analyzed by fluorescence-activated cell sorting (FACS) for dendritic cells, monocytes, and lymphocyte cell surface markers (data not shown). Phenotypically, bone marrow-derived dendritic cells expressed abundant MHC class II, CD80, CD86, CD40, CD11b, DEC 205, and CD11c and lacked CD3 or B220 (T or B cell markers).

Flow cytometry
For flow cytometric analysis, single cell suspensions were washed twice with PBS containing 5% FCS (Life Technologies, Inc.) and 0.1% sodium azide (washing solution). Nonspecific binding was blocked by incubating cells with PBS containing 5% FCS for 30 min at 4 C. Approximately 1–2 x 105 cells were aliquoted in 6-ml polypropylene tubes and incubated with FITC-labeled isotype control antibody or FITC-labeled anti-CD40 mAb for 30 min at 4 C in the dark. The cells were washed twice and fixed in 0.1–0.2 ml of PBS containing 1% paraformaldehyde and 5 mM EDTA before analysis. Flow cytometry was performed using a FACSCalibur with CELLQuest analysis software (Becton Dickinson and Co., San Jose, CA; Ref. 41).

RT-PCR for CD40 and CD40L
Total RNA was isolated from cells using RNAzol B (Life Technologies, Inc.) according to manufacturer’s instructions. cDNA was synthesized from 5 µg of total RNA in a 20-µl reaction mixture containing 1x first stand buffer, 500 µM deoxynucleoside triphosphates, 10 mM dithiothreitol, 500 ng oligo (dT) 12–18 primer, and 200 U Superscript II reverse transcriptase (Life Technologies, Inc., Baltimore, MD). One microliter of cDNA was amplified in a 50-µl PCR containing 1x PCR buffer (Fisher Scientific, Pittsburgh, PA), 200 nM 5' and 3' primer, 200 µM deoxynucleoside triphosphate mixture, 2 mM MgCl2, and 2.5 U Taq DNA polymerase (Life Technologies, Inc.). The following primers were used for cDNA amplification: CD40 sense, 5'-CAT CAC GAC AGG AAT GAC CAG-3'; antisense, 5'-CAT AGA AGA TTG GAT AAG GTC-3'; CD40L sense, 5'-CAG ACT GCT GCT CGC AAA GCT-3'; antisense, 5'-GAT TCG CTG TCA CCA GCA CAG-3'. Amplifications were performed in a DNA thermal cycler (Perkin-Elmer Cetus, Emeryville, CA) for 25–40 cycles using the following reaction profile: 94 C for 45 sec, 58 C for CD40 or 54 C for CD40L for 30 sec, and 72 C for 45 sec. Aliquots of PCR products were run on 1.5% agarose gels and visualized by UV transillumination. Controls included a mouse spleen cDNA (positive for mCD40 and CD40L) and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase.

Mixed lymphocyte reaction (MLR)
The ability of MLO-Y4 cells to act as stimulators was analyzed in an allogeneic MLR. Varying concentrations of irradiated C57BL/6 bone marrow-derived dendritic cells or MLO-Y4 cells were treated with TNF{alpha} (10 ng/ml) or lipopolysaccharide (10 m{gamma}/ml) and cultured in triplicate with purified T lymphocytes (2 x 105 cells/well) derived from the spleens of BALB/c mice using mouse CD3 T cell enrichment column (R&D Systems; Ref. 40). Cells were pulsed with 0.5 µCi tritiated thymidine (NEN Life Science Products, Boston, MA) in the last 8 h of a 72-h coculture (40, 41). The cells were harvested on glass fiber filters (Pharmacia, Uppsala, Sweden) and counted on a {gamma}-scintillation counter (LKB Wallac Oy, Turku, Finland).

Cell treatment
The MLO-Y4 cells were cultured in 2.5% FBS/2.5% calf serum, and the osteoblastic cells in 10% FBS in {alpha}-MEM, 5% CO2 at 37 C. MLO-Y4, OCT-1, and MC3T3-E1 cells were pretreated with CD40L (0.5–1.5 µg/ml) for 15 min, followed by treatment with either dexamethasone (10-6 M), TNF{alpha} (1 ng/ml), or etoposide (50 µM) for 6 h. Previous studies established that this is the earliest time point at which apoptosis can be detected, as assessed by changes in nuclear morphology and membrane permeability (16, 42). The 0.5- to 1.5-µg/ml dose was chosen because 0.1–3 µg/ml was previously shown to be effective in inducing B cells to proliferate without costimulation and to rescue B cell apoptosis (43).

Assays for cell death/apoptosis
Trypan blue exclusion.
Staining of cultured cells with vital dyes such as trypan blue is a commonly used approach to quantify cell death. This assay is based on the premise that viable cells with intact membranes will exclude the dye. The percentage of trypan blue-positive cells in each culture condition was used to calculate cell survival (15). Nonadherent cells were combined with adherent cells released from the culture dish using trypsin-EDTA, resuspended in medium containing serum, and collected by centrifugation. Subsequently, 0.04% trypan blue was added, and the percentage of cells exhibiting both nuclear and cytoplasmic staining was determined using a hemocytometer. At least 100 cells per condition were counted. Apoptosis was confirmed by additional analyses as shown below. In previous studies, the percentage of apoptotic cells determined by trypan blue staining corresponded to that determined by terminal uridine deoxynucleotidyl nick end labeling and was associated with increases in caspase-3 activity (16).

Nuclear morphology.
During apoptosis, the nucleus becomes shrunken and pyknotic as a result of DNA degradation. The terminal stage of apoptosis is characterized by chromatin condensation along the nuclear margin that also coincides with DNA cleavage into small fragments. Visualization of morphological features of apoptosis in the nucleus of the cells, such as chromatin condensation and nuclear fragmentation, was determined in this assay. MLO-Y4 cells were stably transduced with retroviral vector carrying the green fluorescent protein (GFP) cDNA with a nuclear localization sequence designed to target the GFP to the nucleus. MLO-Y4 cells stably transduced with nuclear GFP were fixed in neutral buffer formalin for 8 min, and apoptosis was assessed by enumerating cells exhibiting chromatin condensation and nuclear fragmentation under a fluorescent microscope. At least 500 cells from fields selected by systematic random sampling were examined for each experimental condition (16, 42).

FACS staining for annexin V/PI.
In early stages of apoptosis, cells loose membrane asymmetry and translocate the membrane phospholipid, phosphatidylserine, to the outer leaflet of the plasma membrane where it can be detected by its high-affinity binding to annexin V (44). Prolonged apoptosis can cause cell death, and these nonviable cells stain positive for the vital dye, PI, as well as annexin V. Apoptotic cells stain annexin V positive and PI negative. The staining protocol includes washing single cell suspensions twice with PBS and resuspension in 1x binding buffer [0.01 M HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2] at a concentration of 1 x 106 cells/ml. The cells are aliquoted (100 µl) into polypropylene tubes and incubated with annexin V FITC (5 µl) and 50 µg/ml of PI (10 µl) for 15 min at 4 C in the dark and analyzed by flow cytometry within 1 h. Flow cytometric analysis was performed on a FACSCalibur using CELLQuest software (Becton Dickinson and Co., Mountain View, CA) by analyzing 5–10 x 104 cells per sample using forward and side scatter gates to include late apoptotic cells.

Statistical analyses
Data were analyzed with the one-way ANOVA using the Tukey’s multiple comparison or Student’s-Newman-Keuls’ multiple comparison post hoc test. The effect of CD40L on the proportion of MLO-Y4 cells stably transduced with GFP exhibiting chromatin condensation and/or nuclear fragmentation was also analyzed using exact {chi}2, adjusting the P values depending on the number of comparisons performed using the Bonferroni correction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Primary osteoblasts, OCT-1 cells, and MLO-Y4 cells express CD40 on their surface. MLO-Y4 cells share few phenotypic/cell surface markers with long-lived antigen-presenting immune cells such as B lymphocytes and dendritic cells.
Osteocyte-like MLO-Y4 cells express extensive, complex dendritic processes that are also a phenotypic hallmark of dendritic cells. Their morphology was compared with murine primary bone marrow dendritic cells (Fig. 1Go). The bone marrow-derived dendritic cells do not express as extensive a dendritic network as the MLO-Y4 cells (Fig. 1Go, A and B). We then sought to determine whether the murine osteocyte-like cell line shared any cell surface markers with the murine dendritic cells. Analysis of MLO-Y4 cells by FACS revealed that they did not express CD45, a marker present in abundance on all hematopoietic cells. Markers for T and B lymphocytes (CD3 and B220, respectively), granulocytes (Gr-1), monocytes/macrophages (CD11b), and dendritic cells (DEC-205, CD11c, MHC class II, CD86, and CD80) were also absent (Table 1Go). However, the MLO-Y4 cells did express abundant MHC class I molecules that are expressed by all nucleated cells in the body (Fig. 2AGo), but lacked MHC class II.



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  		Figure 1. Morphological appearance of osteocyte-like MLO-Y4 cells (A) and marrow-derived dendritic cells (B) in 7-d culture. Magnification, x200.

 

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  		Table 1. Cell surface marker expression profile of osteocyte-like MLO-Y4 cells

 


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  		Figure 2. A, Cell surface expression of CD40 and MHC class I antigen on MLO-Y4 cells. B, CD40 expression in primary osteoblasts (PRI OBI), osteoblast cell lines (MC3T3, OCT-1, 2T3), and the osteocyte cell line (MLO-Y4) by flow cytometry. The data are presented as histograms of cell number on the y-axis plotted against log fluorescence intensity on the x-axis. The top panels show the background staining with the isotype control antibody for each cell line, and the bottom panels depict the shift in fluorescence with the mAb specific for either CD40 or MHC class I.

 
Bone cells share survival/death TNF receptor/ligands with immune cells but lack CD40L
Several members of the TNF receptor/ligand family have been shown to play an important role in immune cell (especially dendritic cell and B lymphocyte) survival and apoptosis. Primary osteoblasts and the osteoblast cell lines MC-3T3-E1, 2T3, and OCT-1 also express CD40 (Fig. 2BGo). In the FACS analysis, more than 85–90% of the cells stained positive for CD40. The histograms for the MLO-Y4 cells as well as the primary osteoblasts showed two peaks on the x-axis, suggesting that expression of CD40 on MLO-Y4 cells and primary osteoblasts is heterogeneous. Based on the intensity of fluorescence, these cells can be divided into two groups. Approximately 60–70% of the cells express low levels of CD40 (first peak), and 25–36% of the cells express higher fluorescence (second peak). This heterogeneity was also observed using fluorescent microscopy in which some cells were extremely bright, whereas the remainder were faintly fluorescent (data not shown).

The MLO-Y4 cells express abundant CD40, but lack CD40L by FACS analysis (data not shown). RT-PCR for CD40 showed abundant expression in primary osteoblasts, the OCT-1 osteoblast cells, and the MLO-Y4 osteocyte cells, whereas no bands were detectable for CD40L, although abundant in the mouse spleen cell control (Fig. 3Go).



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  		Figure 3. Expression of mRNA for CD40 but not CD40L in bone cells. The top panel shows CD40 mRNA expression in primary osteoblasts, OCT-1, and MLO-Y4 cells, compared with the mouse spleen as a positive control. The bone cells do not appear to express CD40L as shown in the middle panel. The bottom panel is the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene control.

 
MLO-Y4 cells do not share MLR-stimulating function with dendritic cells
Both the mesenchymal-derived osteoblasts that differentiate into mature osteocytes and the hematopoietic progenitor cells that differentiate into dendritic cells originate in the bone marrow (17). The long complex dendritic processes and abundant expression of gap junction protein, connexin 43, suggest that the MLO-Y4 cells have features important for cell to cell communication. Dendritic cells by their very name have dendritic processes and communicate with the adaptive arm of immune cells. They process foreign proteins into small peptides and present these fragments in the groove of the MHC complex that can then be recognized by lymphocytes. Dendritic cell-mediated T cell proliferation was compared with that of MLO-Y4 cells in an MLR. In contrast to dendritic cells, both unstimulated MLO-Y4 cells and MLO-Y4 cells stimulated with TNF{alpha} or lipopolysaccharide (12 h) were unable to induce T cell proliferation (Fig. 4Go, A and B).



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  		Figure 4. MLO-Y4 cells do not share MLR-stimulating function with dendritic cells. Unstimulated (A) and stimulated (B) MLO-Y4 cells were unable to induce T lymphocyte proliferation as measured by an increase in thymidine incorporation. Increasing numbers of irradiated cells, either dendritic or MLO-Y4 cells, were cultured with 2 x 105 purified T cells in quadruplicate 96-well plates for 72 h, and proliferation was determined by thymidine incorporation as described in Materials and Methods (A). MLO-Y4 cells were stimulated with TNF{alpha} (10 ng/ml) or lipopolysaccharide (10 µg/ml). After irradiation, 1 x 104 MLO-Y4 cells were cultured with 2 x 105 purified T cells and proliferation assessed (B).

 
CD40L prevents glucocorticoid and TNF{alpha}-induced apoptosis in osteocytes and osteoblasts
As cells undergo apoptosis, there are many processes occurring simultaneously, and an assay may measure only one critical component of the apoptotic process. Hence, we evaluated the effects of CD40L on apoptosis-inducing agents by three different techniques. Trypan blue exclusion was used as a measure of cell death in MLO-Y4 cells pretreated with CD40L, followed by dexamethasone, TNF{alpha}, or etoposide (Table 2Go and Fig. 5AGo). The percentage of apoptotic cells was significantly lower in cells that had been pretreated with CD40L independent of the proapoptotic agent used. Consistent with these results, cultures of MLO-Y4 cells stably transfected with nuclear GFP pretreated with CD40L contained a significantly lower percentage of cells exhibiting nuclear fragmentation and/or chromatin condensation after treatment with dexamethasone, TNF{alpha}, or etoposide (Table 2Go and Fig. 5BGo). Similar to the results obtained with the above two methods, the percentage of apoptotic cells identified by annexin V staining was lower in the cells pretreated with CD40L followed by dexamethasone or TNF{alpha}. The data from these different apoptosis assays are summarized in Table 2Go. Like the MLO-Y4 osteocyte cells, CD40L inhibited apoptosis induced by either dexamethasone or TNF{alpha} in osteoblast cells, OCT-1, and MC3T3-E1 (Table 3Go).


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  		Table 2. Three different apoptosis assays showing CD40L blocks apoptosis in osteocytes

 


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  		Figure 5. Effect of CD40L on apoptosis induced by different agents on the MLO-Y4 osteocytic cell line as determined by trypan blue uptake (A) and nuclear fragmentation (B). Cells were pretreated with CD40L (0.5–1.5 µg/ml) followed by dexamethasone (10-6 M), TNF{alpha} (1 ng/ml), or etoposide (50 µM). In each treatment group, the percentage of apoptotic cells was significantly lower in cells that had been pretreated with CD40L. A, In the trypan blue uptake experiment, * represents significantly different from controls using one-way ANOVA, P < 0.05. B, In the nuclear fragmentation experiment, * represents significantly different from controls, P < 0.017; # represents significantly different from the proapoptotic agent alone using {chi}2 test, P < 0.025. Controls included no treatment and CD40L alone.

 

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  		Table 3. CD40L blocks apoptosis in osteoblasts

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here, three novel findings are described. First, CD40, a member of the TNF receptor ligand family, is expressed abundantly in bone cells, namely primary osteoblasts derived from mouse calvaria, osteoblast cell lines, and the osteocyte cell line, MLO-Y4. Second, the ligation of CD40 by CD40L inhibits dexamethasone, etoposide, or TNF{alpha}-induced apoptosis in MLO-Y4 osteocyte cells and in osteoblast cells. Thus, prevention of osteoblast/osteocyte cell death by CD40L may reduce or abrogate the effects of glucocorticoids/steroids on bone or the effects of a lack of estrogen on bone loss. Third, the MLO-Y4 osteocyte-like cells, although dendritic and expressing CD40, do not function as or express all of the markers defining dendritic cells. The lack of MLR-stimulating activity of MLO-Y4 cells is consistent with the absence of expression of class II MHC antigen. The role of CD40, a member of the TNF ligand family, in bone biology has not previously been described.

An important balance must exist between bone and the immune system for bone health. It is not clear what role dendritic cells may play in bone remodeling, although it is known that both B and T cells can play a role in either bone resorption or bone formation. T cells can support or inhibit osteoclast formation, depending on the state of activation (45). An example of an effect of the immune system on bone is autoimmune arthritis, in which increased bone resorption leads to joint destruction (46, 47). Much of the interaction between the immune system and bone takes place through soluble factors, the cytokines. Many of these factors and their receptors were initially discovered in immune cells and have subsequently been identified in bone cells (46). These cytokines can function as either apoptotic or antiapoptotic agents depending on the immune cell or bone cell type. RANKL and TNF{alpha} are antiapoptotic factors for dendritic cells and osteoclasts, whereas TNF{alpha} is an apoptotic factor for osteoblasts and osteocytes. From these initial observations, it might be concluded that dendritic cells share similar properties with osteoclasts; however, our observations using CD40L show that dendritic cells can also share properties with osteoblasts/osteocytes.

CD40L induces apoptosis in transformed or cancer cells but has the opposite effect on normal dendritic cells (48). CD40L is a promising anticancer agent because of this property. Thus, this receptor/ligand signaling pathway may promote cell survival or cell death depending not only on cell type or the stage of differentiation but also on state of transformation. The specificity of these effects could be due to the combination, presence, or absence of specific transcription factors. In our present studies, CD40L clearly prevented apoptosis due to three very different agents, i.e. a steroid, a cytokine, and an apoptosis-inducing chemical compound. All three are purported to work by different pathways through different receptors to induce apoptosis. Therefore, CD40L may support viability of osteoblasts and osteocytes against a wide array or variety of apoptotic factors independent of signaling or transcriptional mechanisms.

The potential role/function of CD40 in bone may be hypothesized from its previously known functions in other cell types such as immune cells. First, one potential function of CD40L in bone could be as a survival factor for bone cells, specifically the osteocyte and osteoblast, because CD40 is a survival factor for dendritic cells. The present studies support this hypothesis. In addition to osteoporosis, during the bone remodeling that occurs with growth, osteocyte cell death is observed (49). CD40 may play a role in bone growth because osteoblasts differentiate into osteocytes. Prevention of osteocyte cell death during growth may increase bone mass or alter skeletal patterning. Second, CD40 may play a role in bone formation and modeling by inducing matrix degrading enzymes such as metalloproteinases, as it does in human vascular smooth muscle cells (50). Enzymes induced by CD40 include interstitial collagenase [matrix metalloproteinase (MMP)-1], stromelysin (MMP-3), gelatinase B (MMP-9), and gelatinase A (MMP-2). Mice expressing a mutated collagen, resistant to digestion by collagenases, have increased deposition of woven bone and other bony defects (51). Therefore, CD40 may regulate production of these enzymes by osteoblasts and osteocytes. Finally, CD40 may have a potential role in osteoclast formation and viability because the molecule is related to TRANCE/RANKL, a molecule necessary for osteoclast formation (23). It will be important to determine the effects of CD40L on osteoclast apoptosis in which it would be predicted to enhance osteoclast cell death. This is based on the observation that antiapoptotic factors for osteoclasts (TNF{alpha}) are often apoptotic factors for osteoblasts and apoptotic factors for osteoclasts (estrogen, TGFß) are often antiapoptotic factors for osteoblasts. The present studies were formulated and based on the first hypothesis, but the next three remain open for investigation.

The TNF receptor/ligand signaling pathway coordinates a wide range of biological responses such as apoptosis, differentiation, and proliferation (52). Binding of members of the TNF receptor family with its ligands results in the activation of several signaling pathways that include caspases, NF-{kappa}B transcription factor, and the MAPK pathway. Depending on the downstream signaling events, this may result in cell survival and proliferation, cell differentiation, or cell death. For example, TRANCE (RANKL) enhances dendritic cell survival and T cell proliferation by up-regulating Bcl-Xl expression (23), whereas Fas ligand, TNF{alpha} and, CD30 ligand play an important role in apoptosis of T lymphocytes after T cell receptor stimulation (5, 23, 25). It will be important to determine the signaling mechanisms used by CD40/CD40L in bone cells to prevent apoptosis.

It will also be important to determine the source of CD40L in the bone microenvironment. Recently, it has been shown that activation of CD40 in B cells and dendritic cells will increase osteoprotegerin, an inhibitor of osteoclast formation (53). Osteoprotegerin has been shown to play a critical role in bone formation. Mice overexpressing this factor have increased bone density, whereas mice lacking the gene for this factor express an osteoporotic phenotype (54). Soluble osteoprotegerin prevents bone loss and bone metastasis. Present studies are focusing on determining the source of ligand in the bone, whether from other bone cells, vascular, or immune cells, and whether activation of CD40 increases osteoprotegerin expression in addition to enhancing viability of osteoblasts and osteocytes.


   Footnotes
 
This work was supported by NIH Grants AR-46798 (to L.F.B. and S.Z.), KO2-AR-02127 (to T.B. and L.I.P.), AI-48644 (to S.S.A. and F.J.), and a Veterans Administration Career Development grant (to S.S.A.).

Abbreviations: CD40L, CD40 ligand; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; mAb, monoclonal antibodies; MLR, mixed lymphocyte reaction; MMP, matrix metalloproteinase; MYO-Y4, murine long bone osteocyte-Y4; NF-{kappa}B, nuclear factor {kappa}B; PI, propidium iodide; RANKL, receptor activator of NF-{kappa}B ligand; TRANCE, TNF-related activation associated cytokine; TRAF, TNF receptor-associated proteins.

Received October 30, 2002.

Accepted for publication January 8, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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