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Endocrinology Vol. 139, No. 10 4353-4363
Copyright © 1998 by The Endocrine Society

ARTICLES

Human Osteoclasts and Osteoclast-Like Cells Synthesize and Release High Basal and Inflammatory Stimulated Levels of the Potent Chemokine Interleukin-81

Linda Rothe, Patricia Collin-Osdoby, Yan Chen, Teresa Sunyer, Lala Chaudhary, Alfie Tsay, Steven Goldring, Louis Avioli and Philip Osdoby

Department of Biology (L.R., P.C.-O., Y.C., T.S., P.O.) and Division of Bone and Mineral Research (P.C.-O., Y.C., L.C., L.A., P.O.), Washington University, St. Louis, Missouri 63130; and the Department of Rheumatology (A.T., S.G.), Harvard Institutes of Medicine, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Dr. Philip Osdoby, Department of Biology, Box 1229, Washington University, St. Louis, Missouri 63130. E-mail: osdoby{at}biodec.wustl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemokines, including interleukin-8 (IL-8), function as key mediators in diverse inflammatory disorders via promoting the recruitment, proliferation, and activation of vascular and immune cells. IL-8 levels are elevated in inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, osteomyelitis, and periodontal disease, that also exhibit progressive bone loss. Therefore, it is possible that IL-8 contributes to the osteopenia associated with these pathological conditions. Although macrophages, neutrophils, and endothelial cells are considered the primary sources of inflammation-induced IL-8 increases, we report here for the first time that human bone marrow-derived osteoclast-like cells (hOCL) as well as authentic bone-resorbing human osteoclasts (hOC) isolated from osteoporotic femoral heads express messenger RNA (mRNA) for IL-8 and secrete high levels of IL-8 during culture. Basal IL-8 release by cultured hOC or hOCL was orders of magnitude greater than the release of the proinflammatory cytokines IL-1ß, IL-6, and tumor necrosis factor-{alpha}. At a cellular level, in situ hybridization analysis revealed that IL-8 mRNA was expressed in resorbing hOC of rheumatoid arthritic pannus and was substantially greater than that expressed in hOC of noninflammatory giant cell tumor of bone tissue. Therefore, the potential inflammation-mediated induction of IL-8 was directly assessed using cultured hOCL. IL-8 release was stimulated by proinflammatory signals (IL-1{alpha}, tumor necrosis factor-{alpha}, lipopolysaccharide, or phorbol 12-myristate 13-acetate), unaffected by various other osteotropic modulators (transforming growth factor-ß1 and -ß3, IL-6, 17ß-estradiol, or calcitonin) and was decreased by interferon-{gamma}, vitamin D3, and the antiinflammatory glucocorticoid dexamethasone. Changes in IL-8 secretion were paralleled by corresponding changes in IL-8 mRNA steady state levels. We conclude that hOC and hOCL synthesize and secrete high constitutive and inflammation-stimulated levels of the chemokine IL-8. Consequently, hOC-derived IL-8 could act as an important regulatory signal for bone, vascular, and immune cell recruitment and activation during normal and pathological bone remodeling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERLEUKIN-8 (IL-8) is the prototypical member of a superfamily of small (8–10 kDa), inducible, secreted chemoattractant cytokines (chemokines) that were originally discovered as monocyte-derived factors capable of attracting and activating neutrophils (1, 2, 3, 4). Many cell types are now known to synthesize and release IL-8 (and other chemokines) in response to injury, infection, inflammation, or various pathological conditions (2, 3, 4, 5). IL-8 production is induced by proinflammatory cytokines [IL-1, tumor necrosis factor-{alpha} (TNF{alpha}), or granulocyte-macrophage colony-stimulating factor] or other immune stimuli [bacterial lipopolysaccharide (LPS), lectins, and protein kinase C activation]. The precursor protein (99 amino acids) undergoes cell type-dependent sequential N-terminal proteolytic processing to yield products (69–79 amino acids) of varying bioactivities (6). In addition to serving as a potent chemoattractant activator of neutrophils, IL-8 exerts other diverse proinflammatory or physiological effects on target cells, including the stimulation of hematopoiesis, angiogenesis, mitogenesis, bioactive lipid formation, protease activation, the release of lysosomal enzymes, reactive oxygen species, cytokines, and nitric oxide, and rapid changes in the expression or conformation of cell surface adhesion proteins (e.g. ß-integrins and Mac-1), F-actin, and cell shape (2, 3, 4, 5, 6, 7, 8, 9, 10, 11). Such biological actions are typically mediated via elevated intracellular Ca2+ levels (12) that occur following IL-8 ligand binding to specific cell surface receptors that belong to the superfamily of seven-transmembrane spanning G protein-coupled receptors (2, 3, 4, 5, 6, 7, 8, 9, 13). Consistent with its proinflammatory actions in vitro, IL-8 levels are markedly elevated in various inflammatory disorders, such as rheumatoid arthritis, osteoarthritis, periodontal disease, psoriatic lesions, pulmonary diseases, endotoxemia and sepsis, gastrointestinal inflammation, and immune complex glomerulonephritis (2, 7, 8, 9). Whereas ip or sc injections of IL-8 cause neutrophil migration, infiltration, and activation at such sites (2, 7, 8, 9), neutralizing antibodies to IL-8 conversely suppress acute inflammatory reactions in various inflammatory conditions (8, 14). These and many other studies have shown IL-8 to be a major mediator involved in biological inflammatory responses.

Bone loss is a typical hallmark of many inflammatory conditions and is thought to be a consequence of both reduced osteoblast bone formation and increased osteoclast (OC) bone resorption. Recently, we showed that IL-8 (0.1–1.0 nM) directly inhibits osteoblast characteristics important for their bone formative function (15, 16). Therefore, a local rise in IL-8 levels could presumably contribute to regional bone loss through suppressive effects on osteoblasts. At higher concentrations (>=10 nM), IL-8 also partially inhibits the overall bone-resorptive activity of isolated rat and chick OC; however, this appears to reflect IL-8-evoked increases in OC motility to new sites of resorption rather than actual declines in the resorptive capability of individual OC per se (15, 17, 18). Formerly, human osteoblast-like cells (hOBL) were shown to also produce this chemokine, the levels of which were elevated by inflammatory cytokine stimulation and conversely suppressed by the antiinflammatory glucocorticoid dexamethasone (19). However, aside from these initial studies, little is known about the role of IL-8 in bone physiology, specifically in relation to either osteoblast or OC development or function. During the course of studies employing human OC-like cells (hOCL), which exhibit most of the attributes of bone-resorptive OC (20), we discovered that high levels of IL-8 were released into the culture medium. This prompted us to ask whether human OC (hOC) might also produce this potent intercellular signal molecule, a question that had not been addressed before the current work. Over the past few years, OC have been increasingly recognized as capable of producing a variety of autocrine/paracrine-acting bone-active cytokines, free radicals, matrix proteins, and other signal molecules. Thus, we explored whether bone-resorbing hOC and related hOCL expressed messenger RNA (mRNA) for IL-8, released measurable levels of this chemokine, and exhibited altered IL-8 production in response to inflammatory stimuli. Moreover, IL-8 mRNA expression levels in resorbing hOC were assessed by in situ hybridization in inflammatory and noninflammatory human bone tissues to learn whether IL-8 levels were correlated with conditions of localized osteopenia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
hOC and hOCL
Authentic bone-resorptive hOC (<=107) were isolated from femoral heads discarded during hip replacement surgery on osteoporotic patients or from segments of bone removed from sites of implant loosening during resection (21). Briefly, the bone was cut into small pieces and trypsin/collagenase treated with shaking, and the released cells were enriched (to >40% and >80% on a per cell and a per nuclear basis, respectively) for hOC by Percoll fractionation or serum sedimentation. Highly purified (~90% on a per cell basis, >98% on a per nuclear basis) hOC preparations were obtained by immunomagnetic sorting using the specific anti-OC monoclonal antibody (Mab) 121F (22). hOC preparations were cultured in OC medium [phenol red-free medium 199 Earle’s salts with 8.3 mM NaHCO3, 100 mM HEPES (pH 6.8), 5% FBS (Life Technologies, Gaithersburg, MD), and 2.5% antibiotic/antimycotic] at 37 C in a 95% air-5% CO2 moist environment. The cells expressed characteristic OC features: high tartrate-resistant acid phosphatase activity, vitronectin receptor ({alpha}Vß3 integrin), 121F Mab-reactive antigen, mRNAs for calcitonin receptor, cathepsin K/O, and carbonic anhydrase II, and resorption pit formation during culture on ivory or devitalized bovine cortical bone slices (21). In vitro formed hOCL were generated using bone marrow from discarded surgical specimens of human rib bone, the mononuclear cells were Ficoll-Hypaque (Pharmacia, Piscataway, NJ) separated, and the cells were resuspended in {alpha}MEM (Life Technologies) with 10% FBS and 2% antibiotic/antimycotic for culture at 40 x 106 cells/100-mm dish (21, 22). On day 6, nonadherent cells were replated in medium with 10 nM 1,25-dihydroxyvitamin D3 and thereafter refed every 3 days with medium containing 25% conditioned medium (CM) from UMR 106-01 OBL to further OC-like differentiation (22, 23). After 2–3 weeks, stromal cells were selectively removed by brief trypsinization, and hOCL were detached to replate into 24-well dishes at 0.8 x 105 cells/well (21). After 48 h, hOCL were switched to phenol red-free medium, modulators were given in fresh medium 1–2 days later, and CM was collected, spun, and stored at -80 C. These multinucleated hOCL express the key OC phenotypic features listed above for hOC, but form few, if any, resorption pits in culture on ivory or bone slices (20, 21).

Human bone marrow stromal (hBMS) and hOBL cells
hBMS were obtained as the first adherent cell population of the initial plating of human marrow mononuclear cells (above) based on the report of Cheng et al. (24). Briefly, after removal of the day 6 nonadherent cells, the adherent cells were cultured to confluence, trypsin detached, and replated at 2 x 105 cells/100-mm dish (21). hBMS were refed before 48-h CM collection from near-confluent cultures. For hOBL differentiation, 10-7 M dexamethasone was added to the proliferating hBMS (day 2) and withdrawn (day 7) before collection of 48-h CM (24).

Modulator treatments
The following reagents were obtained from Sigma Chemical Co. (St. Louis, MO): Escherichia coli LPS serotype 0111:B4 stored at 4 C as a 5 mg/ml stock in Hanks’ Balanced Salt Solution, phorbol 12-myristate 13-acetate (PMA) stored at -80 C as a 8.1 x 10-4 M stock in ethanol, dexamethasone freshly prepared as a 10-3 M stock in ethanol, 17ß-estradiol stored at -20 C as a 3.7 x 10-4 M stock in ethanol, and BSA fraction V (<0.1 ng/ml endotoxin). 1,25-(OH)2D3, a gift from Hoffmann-La Roche (Nutley, NJ), was stored dry at -80 C and reconstituted at 10-3 M in ethanol before use. Salmon calcitonin, also a gift from Hoffmann-La Roche, was stored as 10-3-M aliquots (4411 IU/mg) in acetate solution at -80 C. Human recombinant cytokines [interferon-{gamma} (IFN{gamma}), IL-1{alpha}, IL-6, TNF{alpha}, transforming growth factor-ß1 (TGFß1), and TGFß3] were purchased from R&D Systems (Minneapolis, MN), reconstituted in PBS with 0.1% low endotoxin BSA (and 4 mM HCl for TGFß stocks), and stored as aliquots at -80 C. Modulators were diluted in medium just before their administration to cells, vehicle controls were tested in parallel, and CM was collected after 24 h, spun, and stored at -80 C.

Quantification of cytokine release
Cytokine levels in CM were measured using specific enzyme-linked immunoassay kits (R&D Systems) as recommended. Standard curves were run with every assay. Each modulator was tested in three or more separate culture wells per trial, and two to six independent trials were performed per condition. Corresponding control medium for each trial exhibited insignificant reactivity with each cytokine enzyme-linked immunosorbent assay. Minimum detectable cytokine levels were 3.0 pg/ml IL-8, 0.064 pg/ml IL-6, 0.1 pg/ml IL-1ß, and 0.11 pg/ml TNF{alpha}. Results are expressed as nanograms of cytokine released per ml culture medium or as nanograms of cytokine released per 106 cells. Normalization to cell protein was performed using harvested cells in the bicinchoninic acid protein assay (Pierce, Rockford, IL) with BSA as a standard.

RNA isolation and RT-PCR
Total RNA was extracted using RNA STAT-60 (Tel-Test, Friendswood, TX), first strand complementary DNA (cDNA) synthesized using a cDNA cycle kit (Invitrogen, San Diego, CA), and PCR reactions were performed using specific primer pairs from these locations: 1) 192–212 and 338–317 of GenBank locus HSMDNCF for the 147-bp IL-8 amplicon, 2) 340–359 and 494–475 from locus HUMACP5 for the 155-bp tartrate-resistant acid phosphatase amplicon, 3) 409–426 and 661–642 from locus HUMCALREC for the 252-bp calcitonin receptor amplicon, 4) 3325–3344 and 3764–3747 from locus HUMGAPDHG for the 221-bp glyceraldehyde-3-phosphate dehydrogenase amplicon, and 5) 749–770 and 1093–1071 for the first round, and 800–822 and 1093–1071 for the second round, from locus HUMERMCF for the 249-bp estrogen receptor (ER) amplicon as previously described (25). PCR products were separated in a 1.5% agarose gel and ethidium bromide stained. All were of the expected size for the primers used, and sequencing of select amplicon products, including IL-8 and estrogen receptor, was performed to confirm their identity.

Northern blot analysis
Total RNA (20–25 µg) was separated on denaturing 1% agarose/formaldehyde gels (26), ethidium bromide stained to assess RNA integrity/loading, transferred to nylon membranes (Schleicher and Schuell), fixed by UV cross-linking (UV Stratalinker 2400, Stratagene, La Jolla, CA), and prehybridized, and the blots were hybridized overnight at 42–45 C with a labeled 406-bp human IL-8 cDNA probe (provided by Dr. S. Kunkel, Pathology, University of Michigan Medical School, Ann Arbor, MI) (27) generated by random primer extension using [32P]deoxy-CTP (New England Nuclear, Boston, MA) or by digoxygenin-11-deoxy-UTP labeling (Genius, Boehringer Mannheim, Indianapolis, IN). After hybridization with 106 cpm/ml 32P-labeled probe (see Fig. 7AGo) or 10-10 M digoxygenin-11-deoxy-UTP-labeled probe (see Fig. 7BGo), specific signals were detected by autoradiography or chemiluminescence on XAR-5 film (Eastman Kodak, Rochester, NY). Stripped blots were rehybridized with a labeled cDNA probe for 18S RNA (provided by Dr. J. Lian (26)) for normalization of IL-8 signals. Blots were quantified (sum of the gray area) using a densitometer (Hewlett-Packard Scanjet, Palo Alto, CA) linked to a Leica Quantimet image analysis system (Leica, Deerfield, IL), and the results were expressed in arbitrary densitometric units as relative ratios for IL-8/18S.



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  		Figure 7. Steady state IL-8 mRNA levels are regulated in hOCL by proinflammatory and antiinflammatory stimuli. A, hOCL were cultured in the absence or presence of PMA (0.01 nM) or LPS (0.5 µg/ml) for 8 h, after which RNA was extracted from the cells, separated on agarose gels, and blotted to nylon membranes for Northern analysis of IL-8 mRNA steady state levels. Subsequently, blots were stripped and rehybridized with a probe to 18S ribosomal RNA to normalize specific hybridization signals, and autoradiograms were quantified by densitometric analysis as described in Materials and Methods. Normalized data for ratios of IL-8 mRNA relative to 18S ribosomal RNA are depicted graphically in arbitrary densitometric units, and the corresponding gel profiles for IL-8 and 18S hybridizations are shown below the graph. B, hOCL were cultured in triplicate wells with IL-1{alpha} (0.1 nM) or LPS (10 ng/ml), with or without dexamethasone (10 nM), for 8 h before RNA was extracted from each well and subjected to Northern analysis for IL-8 mRNA steady state levels and 18S normalization of blots. Data are presented as the mean ± SEM of IL-8 mRNA levels relative to 18S ribosomal RNA levels for triplicate wells in comparison with the mean IL-8/18S ratio determined for LPS-treated hOCL (set at 100%).

 
In situ hybridization of human bone tissue sections
Human bone tissue samples from giant cell tumors of bone or individuals with rheumatoid arthritis were fixed in 4% paraformaldehyde (PF) for 2 days, decalcified (<=5 weeks) in 14% EDTA, washed in graded ethanols, transferred to xylenes, and embedded in paraffin, and thin sections (5 µm) were cut and dried onto poly-L-lysine-coated slides (28, 29). Slides were deparaffinized in xylenes, rehydrated in graded ethanols, rinsed (5 min each in 0.85% NaCl and PBS), refixed (4% PF, 20 min), PBS washed, proteinase K treated (20 µg/ml, 20 min), dipped in PBS (5 min) and then in 4% PF (5 min), acetylated in 0.25% acetic anhydride/100 mM triethanolamine, washed (5 min each in PBS and 0.85% NaCl), dehydrated in graded ethanols, and air-dried. Sense and antisense riboprobes were prepared from linearized IL-8 cDNA vector (above) by incubation with T7 or SP6 RNA polymerase and [35S]UTP (New England Nuclear) in the presence of unlabeled nucleotides, 10 mM dithiothreitol, and ribonuclease (RNase) inhibitor (Promega, Madison, WI), and the probes were purified using NucTrap push columns (Stratagene, La Jolla, CA). Probes (5 x 104 cpm/µl) were diluted in hybridization buffer [4 x SSC (standard saline citrate), 50% formamide, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 10% dextran sulfate, 1 x Denhardt’s solution, 0.5 mg/ml total yeast RNA, 50 µg/ml yeast transfer RNA, 100 µg/ml denatured salmon sperm DNA, 100 mM dithiothreitol, 0.1% SDS, and 0.1% sodium thiosulfate], 50 µl were added per slide, a glass coverslip was placed on top, and the slides were incubated (55 C, 16 h), washed (four times, 5 min each time, 25 C, 2 x SSC-10 mM 2-mercaptoethanol-1 mM EDTA), and RNase A treated (20 µg/ml, 30 min; Pharmacia Biotech, Piscataway, NJ). Washed slides (three times, 5 min each time, 60 C, 0.1 x SSC) were dehydrated in graded ammonium acetate-ethanol solutions, air-dried, dipped in Kodak NTB-2 emulsion, drained, air-dried (1 h), and placed in a light-proof container with dessicant at 4 C for 4–8 weeks. Slides were developed in Kodak D-19 developer, fixed, and hematoxylin/eosin counterstained. Controls included sense probes and RNase treatment of tissue sections before hybridization.

Statistical analysis
Data are presented as the mean ± SEM of at least three culture wells in each of two to six independent trials. Differences between treatments were analyzed using one-factor ANOVA. For simultaneous comparisons between multiple treatments, significant differences were determined using the post-ANOVA Bonferroni test. Differences were considered significant for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
High levels of IL-8 are released by hOC and hOCL in culture
During the course of studies designed to examine the possible production of inflammatory cytokines by multinucleated hOCL formed and differentiated in vitro from human bone marrow mononuclear cells, we discovered that such cells released high levels (137 ng/106 cells or 22 ng/ml) of the proinflammatory chemokine IL-8 into the CM (Table 1Go). By comparison, the same CM samples contained much lower basal release levels of the three other key inflammatory cytokines, IL-6 (150-fold), TNF{alpha} (1,000-fold), and IL-1ß (17,000-fold), on either a per cell or a per ml basis in relation to IL-8 (Table 1Go). Whether authentic bone-resorptive hOC also released significant basal amounts of IL-8 was analyzed using hOC that were isolated and partially purified in quantity from human femoral head samples, and subsequently cultured for 24 h in vitro (Table 1Go). Like hOCL, hOC exhibited high levels of IL-8 release (197 ng/106 cells or 48 ng/ml) into the CM, and hOC secretion of IL-8 exceeded the release of TNF{alpha} (by 750-fold), IL-6 (by 25- to 40-fold), and IL-1ß (by 200-fold). Somewhat unexpected was the finding that IL-1ß release was substantially greater (by ~150-fold) in authentic hOC cultures than in hOCL cultures (Table 1Go); possibly this relates to having isolated hOC from osteoporotic bone, whereas marrow cells were obtained from normal human bone for hOCL formation. Otherwise, hOC and hOCL released comparable amounts within the same order of magnitude of IL-8, IL-6, and TNF{alpha}. In contrast to hOC and hOCL, basal IL-8 release by human bone cell populations unrelated to the hematopoietic lineage, such as hBMS and stromal-derived hOBL cells, was notably lower on both a nanograms per ml and per cell basis (Table 2Go). Although IL-8 release from differentiated hOBL was 10-fold greater than that from hBMS osteoprogenitor cells, such levels were still 10-fold below the high constitutive levels of IL-8 released by hOC and hOCL. Consistent with detecting IL-8 protein in the CM, IL-8 mRNA was present in both hOC and hOCL based on RT-PCR analysis (Fig. 1Go). Therefore, both hOC and hOCL express IL-8 mRNA and release high levels of IL-8 into the CM during their culture in vitro.


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  		Table 1. Inflammatory cytokine release from human osteoclasts and osteoclast-like cells

 

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  		Table 2. Differential IL-8 release from human bone marrow cells

 


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  		Figure 1. Human OC and OCL express IL-8 mRNA. RT-PCR amplicons of the expected size (147 bp) were obtained using IL-8 primers and RNA prepared from highly purified, 121F immunomagnetically isolated authentic bone-resorbing hOC or in vitro formed hOCL as described in Materials and Methods. Products were separated on agarose gels and stained with ethidium bromide. Amplicon products excised from gels were used for nucleotide sequencing to confirm that they corresponded to human IL-8 mRNA. Lane 1, Size markers; lane 2, negative control; lane 3, PCR product from hOC; lane 4, PCR product from hOCL.

 
IL-8 expression is localized to hOC of human bone tissue and elevated in rheumatoid arthritis
As other cells (e.g. macrophages and vascular endothelial cells) are also known to produce IL-8, and the hOC preparations do not represent pure cell populations, it was important to establish whether IL-8 production was localized to hOC of bone. Human bone tissue sections prepared from individuals with or without an inflammatory bone disorder were probed by in situ hybridization for IL-8 mRNA expression. Whereas a faint hybridization signal indistinguishable from the control signal (Fig. 2AGo) was seen for hOC in sections from a noninflammatory sample such as human giant cell tumor of bone (Fig. 2Go, B and C), a more pronounced and specific hybridization signal was localized to hOC of rheumatoid arthritic bone that had been similarly processed (Fig. 2Go, E and G). Negligible hybridization signals were obtained using control sense probes on parallel tissue sections from either the rheumatoid arthritic (Fig. 2Go, D and F) or giant cell tumor of bone samples (Fig. 2AGo), or if the sections were pretreated with RNase before hybridization with the IL-8 antisense probe (not shown). Therefore, the IL-8 antisense signal localization to individual resorbing hOC of human bone tissue indicates that hOC synthesize mRNA for IL-8 and, consequently, may contribute to or account for the release of this potent proinflammatory factor in isolated hOC cultures. Moreover, hOC IL-8 mRNA levels in vivo may be significantly elevated in inflammatory bone disorders associated with osteopenia, such as rheumatoid arthritis.



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  		Figure 2. IL-8 mRNA expression detected in resorbing hOC by in situ hybridization analysis of human bone tissues. Sections of human bone tissue from patients diagnosed with giant cell tumors of bone (A–C) or rheumatoid arthritis (D–H) were prepared, subjected to in situ hybridization with a radiolabeled sense or antisense probe for IL-8, autoradiographed, and counterstained with hematoxylin and eosin as described in Materials and Methods. A, Human giant cell tumor of bone section hybridized with a sense probe to IL-8 as a control. Giant cells (arrows) are negative for a hybridization signal. Magnification, x428. B, Little IL-8 antisense hybridization signal, indistinguishable from negative sense hybridization controls, is detectable in multinucleated resorbing hOC (arrows) of giant cell tumor of bone tissue. Magnification, x428. C, Higher magnification of multinucleated hOC (arrow) in human giant cell tumor of bone, demonstrating the low degree of hybridization with IL-8 probe observed in this noninflammatory tissue. Magnification, x685. D and F, Human rheumatoid arthritic pannus section hybridized with a sense probe to IL-8 as a control. Multinucleated hOC (arrows) evidence minimal hybridization signals. Magnification, x385. E and G, Resorbing multinucleated hOC (arrows) exhibit moderate to high hybridization signals with the IL-8 antisense probe. Magnification, x550. Control hybridization sections pretreated with RNase also yielded negligible signals in each of these bone tissue types (not shown).

 
Inflammatory stimuli further increase the high levels of IL-8 released by hOCL
The in situ findings suggested that IL-8 mRNA expression by hOC of bone may be up-regulated at sites of inflammation, perhaps in response to locally elevated concentrations of proinflammatory cytokines such as IL-1 and TNF{alpha}. Because hOCL express multiple OC characteristics and release IL-8 at levels comparable to hOC, and larger numbers of hOCL are more readily available and of greater purity than isolated hOC, hOCL were employed in further studies to investigate the potential regulation of IL-8 production in response to inflammatory signals. The proinflammatory cytokines IL-1{alpha} and TNF{alpha} each significantly and dose dependently raised the levels of IL-8 released into the CM beyond the already high basal level exhibited by hOCL (Fig. 3Go, A and B), and such rises were initially detectable as early as 6 h after stimulation (not shown). Bacterial LPS generated a striking rise in the IL-8 release by these cells (Fig. 3CGo), as did the phorbol ester PMA, which directly activates protein kinase C, an intracellular signal commonly triggered by inflammatory stimuli (Fig. 3DGo).



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  		Figure 3. Inflammatory stimulation of IL-8 release by cultured hOCL. Cultures of hOCL were treated with increasing concentrations of IL-1{alpha} (A), TNF{alpha} (B), LPS (C), or PMA (D) for 24 h, and the levels of IL-8 secreted into the culture medium were quantified as described in Materials and Methods. Data were obtained from at least three independent trials for each condition, corrected for nonspecific backgrounds, and the mean ± SEM are presented as a percentage of the IL-8 released by control cultures (100% = 94 ± 5 ng IL-8 released/ml/mg cell protein). Significant differences from control cultures are denoted by **** (P < 0.001).

 
Noninflammatory cytokines and systemic hormones do not stimulate hOCL IL-8 release
Unlike the proinflammatory cytokines IL-1{alpha} or TNF{alpha}, other bone-active cytokines such as IL-6 (10-8–10-11 M), TGFß1 (10-8–10-12 M), or TGFß3 (10-8–10-12 M) did not stimulate IL-8 release from hOCL (not shown), whereas IFN{gamma} (50, 100, or 200 U/ml) reduced constitutive IL-8 release from hOCL (by 60 ± 2%, 62 ± 3%, and 63 ± 4%, respectively; all P < 0.005). Neither of the antiresorptive osteotropic hormones, 17ß-estradiol (10-6–10-10 M) or calcitonin (10-6 to 10-10 M), altered the basal level of IL-8 secretion from hOCL (not shown), although mRNA for receptors of both of these hormones were present in cultured hOCL (Fig. 4Go). Small, but significant, reductions in basal IL-8 release by hOCL occurred after treatment with 10-6–10-8 M 1,25-(OH)2D3 (Fig. 5AGo). However, a more marked and dose-dependent suppression of basal IL-8 release by hOCL was achieved in response to 10-9 or 10-8 M of the antiinflammatory glucocorticoid dexamethasone (Table 3Go), which was not further reduced at a concentration of 10-7 M of this osteotropic hormone (20 ± 4% of control; P < 0.001).



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  		Figure 4. Human OCL mRNA expression of characteristic OC markers. RT-PCR amplicons of the expected sizes were obtained using specific primers and RNA prepared from in vitro formed hOCL as described in Materials and Methods. Products were separated on agarose gels and stained with ethidium bromide. Amplicon products excised from gels were used for nucleotide sequencing to confirm that they corresponded to expected products. Lanes 2 and 6, Size markers; lane 1, estrogen receptor-{alpha} (249 bp); lane 3, glyceraldehyde-3-phosphate dehydrogenase (221 bp); lane 4, tartrate-resistant acid phosphatase (155 bp); lane 5, calcitonin receptor (252 bp). Similar results were obtained in at least two independent trials for each marker.

 


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  		Figure 5. Vitamin D3 inhibition of constitutive and inflammation-stimulated IL-8 release from cultured hOCL. Cultures of hOCL were treated with increasing concentrations of vitamin D3 in the absence (A) or presence (B) of IL-1{alpha} (0.1 nM) for 24 h, after which the levels of IL-8 secreted into the culture medium were quantified as described in Materials and Methods. Data were obtained from at least two independent trials, each performed in triplicate culture wells for each condition. A, IL-8 release is presented as the mean ± SEM nanograms of IL-8 released per ml/mg cell protein. Significant differences from control cultures are indicated (*, P < 0.05; ***, P < 0.005; and ****, P < 0.001). B, IL-8 release is presented as a percentage of the IL-8 secreted by control cultures of hOCL (100% = 100 ± 3 ng IL-8 released/ml·mg cell protein). Significant differences from control cultures (++++, P < 0.001) and significant differences from IL-1{alpha}-stimulated levels (*, P < 0.05, **, P < 0.01, and ***, P < 0.005) are indicated.

 

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  		Table 3. Interdependent regulation of proinflammatory cytokines in human osteoclast-like cells

 
Interdependent regulation of inflammatory cytokine release by hOCL
In addition to the effects of IL-1{alpha} and TNF{alpha} on stimulating IL-8 release from hOCL, IL-1{alpha} significantly and dose dependently increased the hOCL release of both TNF{alpha} and IL-6 (Table 4), and TNF{alpha} dose dependently elevated both IL-1ß and IL-6 release (Table 3Go). However, just as IL-6 treatment of hOCL did not modify IL-8 release, neither did IL-6 alter IL-1ß or TNF{alpha} release from hOCL (Table 3Go). The antiinflammatory glucocorticoid dexamethasone conversely reduced basal IL-8 release concurrent with that of IL-1ß, IL-6, and TNF{alpha} (Table 3Go). Calcitonin (10-10–10-6 M) and 17ß-estradiol (10-10–10-6 M), which had no effects on basal IL-8 production by hOCL, also did not alter IL-1ß or IL-6 release by hOCL (not shown). Thus, complex interdependent regulatory pathways govern the production of these inflammatory-associated bone-active cytokines from hOCL, which might generate autocrine feedback and amplification regulatory circuits.

Cytokine stimulated levels of IL-8 release from hOCL are inhibited by 1,25-(OH)2D3 or by dexamethasone
As 1,25-(OH)2D3 (Fig. 5AGo) and dexamethasone (Table 4) each reduced basal IL-8 release from hOCL, their effects on stimulated IL-8 release were investigated. IL-1{alpha} stimulated IL-8 release was decreased by cotreatment of hOCL with 1,25-(OH)2D3, although such IL-8 levels remained significantly raised over those of unstimulated control cultures in the presence of as much as 10-7 M of this hormone (Fig. 5BGo). The very high levels of IL-8 release stimulated by either LPS (0.1–10 µg/ml) or PMA (10-8–10-6 M) were not reduced by cotreatment of hOCL with 10-8 M 1,25-(OH)2D3 (not shown). Dexamethasone was more potent than 1,25-(OH)2D3 in diminishing both basal (Fig. 6AGo) and IL-1{alpha}-stimulated (Fig. 6BGo) IL-8 release. In addition, dexamethasone potently and dose dependently decreased hOCL release of IL-8 stimulated by either TNF{alpha} (Fig. 6CGo) or LPS (Fig. 6DGo). In each case, 10-7 M dexamethasone was sufficient to return stimulated IL-8 release to levels indistinguishable from control values. In contrast, IL-1{alpha} (10-10 M)-stimulated IL-8 release by hOCL was not altered by cotreatment with calcitonin (10-8 M), 17ß-estradiol (10-8 M), TGFß1 (10-10–10-8 M), or TGFß3 (10-10–10-8 M; not shown).



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  		Figure 6. Dexamethasone inhibition of constitutive and inflammation-stimulated IL-8 release by cultured hOCL. Cultures of hOCL were treated with increasing concentrations of dexamethasone in the presence or absence (A) of 0.1 nM IL-1{alpha} (B), 0.1 nM TNF{alpha} (C), or 0.1 µg/ml LPS (D) for 24 h, and the levels of IL-8 secreted into the culture medium were quantified as described in Materials and Methods. The data shown were obtained from a representative trial performed in triplicate for each condition, which was replicated at least twice with similar results. IL-8 release is presented as a percentage of the IL-8 secreted by control cultures (100% = 94 ± 5 ng IL-8 released/ml·mg cell protein). Significant differences from control cultures are denoted in A by **** (P < 0.001) and in B–D by +++ (P < 0.005) and ++++ (P < 0.001). Significant differences from stimulated levels of IL-8 release in B–D are denoted by *** (P < 0.005) and **** (P < 0.001). Note that at 100 nM dexamethasone, hOCL IL-8 release in the presence of inductive cytokines or LPS is no longer significantly different from that in control unstimulated cultures.

 
Steady state IL-8 mRNA expression levels correlate with IL-8 cytokine release levels by hOCL in response to modulators
Consistent with the large increases in IL-8 cytokine release by hOCL evoked by LPS or PMA, IL-8 mRNA steady state levels in hOCL were also markedly increased more than 2.5-fold in response to these stimuli (Fig. 7AGo). Similarly, steady state IL-8 mRNA levels were elevated by IL-1{alpha} treatment of hOCL (Fig. 7BGo). Conversely, cotreatment with dexamethasone dramatically reduced, by 4- to 6-fold, the elevated IL-8 steady state mRNA levels elicited by either LPS or IL-1{alpha} (Fig. 7BGo). Consistent with its reduction of basal IL-8 release from hOCL (Table 4), dexamethasone also decreased (by 2-fold) the basal IL-8 mRNA steady state levels in hOCL (not shown). These findings were confirmed and extended by RNase protection assays, which also demonstrated that IL-8 mRNA levels were partially elevated by 2 h, peaked within 4–8 h, and thereafter declined by 24 h in response to IL-1 stimulation (not shown). Thus, the regulation of IL-8 cytokine release by hOCL in response to LPS, PMA, IL-1{alpha}, and dexamethasone is reflected in corresponding parallel changes in hOCL steady state expression levels for IL-8 mRNA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, several studies have indicated that human OC may constitutively express mRNA and protein for the inflammatory cytokines IL-6, TNF{alpha}, and IL-1ß in vivo (30); that rabbit OC contain mRNA encoding IL-1ß, IL-6, IL-8, and the chemokine epithelial neutrophil activating peptide-78 (31); and that mouse OC express mRNA for the chemokine macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) in vivo (32). This and other recent evidence suggest that OC may function as both effector and target cells in local autocrine/paracrine regulatory circuits that govern normal and pathological bone remodeling. Here, we report on our detailed studies of the expression, production, and regulation of the proinflammatory chemokine IL-8, in relation to IL-1ß, TNF{alpha}, and IL-6, by isolated authentic bone-resorptive hOC and closely related in vitro formed bone marrow-derived hOCL. The findings have demonstrated for the first time that hOC and hOCL express mRNA for IL-8 and secrete high basal levels of this potent chemokine, which can be further increased by inflammatory conditions or signals. Overall, the similar basal levels (within 1.5- to 4-fold) of IL-8, TNF{alpha}, and IL-6 secreted by cultured hOC and hOCL provide further evidence of the OC-like nature that develops during the in vitro formation of hOCL (20, 22, 33). More importantly, compared with the relatively low basal levels of IL-1ß, TNF{alpha}, and IL-6 produced by cultured hOC and hOCL, IL-8 release was orders of magnitude higher (on a per cell or a per ml basis). In fact, the levels of IL-8 constitutively released by cultured hOC (48 ng/ml; 6 nM) and hOCL (22 ng/ml; 2.75 nM) or released by cultured hOCL in response to inflammatory stimulation by IL-1{alpha} or TNF{alpha} (elevated 2- to 4-fold to 5–11 nM) were similar to those reported for endothelial cells (34, 35, 36) and activated neutrophils or macrophages (37, 38, 39), cells that are considered primary effector cells for mediating physiological and pathophysiological immune-related processes. Thus, IL-8 release by hOC and hOCL was at concentrations known to be biologically effective for physiological cell responses via either of the two known high affinity cell surface IL-8 receptors (CXCR-1 and CXCR-2), each of which exhibits Kd values ranging from 0.1–10 nM in various cell types (40). Although it is possible that other cells present in the hOC preparations may have contributed to the levels of IL-8 release measured, we confirmed that IL-8 mRNA was expressed in highly purified 121F Mab immunomagnetically isolated hOC preparations as well as in highly purified hOCL cultures and showed by in situ hybridization analysis that IL-8 mRNA expression was localized to individual resorbing hOC of human bone tissue. Therefore, hOC potentially represent an additional source of this potent chemoattractant and activating factor in bone, and its regulated release by hOC could exert important paracrine or autocrine modulatory influences on cells within the bone microenvironment, affecting cell recruitment, attachment, differentiation, and physiological function.

Virtually all IL-8-producing cell types respond to inflammatory mediators, especially IL-1{alpha} and TNF{alpha}, with increased IL-8 mRNA expression and IL-8 production (2, 3, 4, 19, 27). Several transcriptional regulatory elements in the promoter region of the human IL-8 gene, including activating protein-1 (AP-1), AP-2, nuclear factor-{kappa}B, nuclear factor-IL-6-C/EBP, hepatocyte nuclear factor-1 (HNF-1), glucocorticoid response element, and IFN regulatory factor-1 sites, mediate these responses (3). In hOCL, as in other cells, both IL-8 mRNA levels and IL-8 release were raised by the inflammatory stimuli PMA, LPS, IL-1{alpha}, and TNF{alpha}, and conversely decreased by the antiinflammatory glucocorticoid dexamethasone. In contrast, IL-6 did not promote hOCL release of IL-8, IL-1ß, or TNF{alpha}, consistent with its general inability to induce such cytokines in a broad range of cell types (41), although in one report IL-6 induced the chemokine monocyte chemoattractant protein-1 in human and rat osteoblasts (42). Although TGFß isoforms have variably had no effect on (2), augmented (43), or inhibited (44) cytokine-stimulated IL-8 production in other cells, and inhibited MIP-1{alpha} production by bone marrow macrophages (45), neither TFGß1 nor TGFß3 altered either basal or IL-1{alpha}-induced IL-8 secretion by hOCL. Whereas 1,25-(OH)2D3 decreased both basal and IL-1{alpha}-stimulated IL-8 release by hOCL, similar to its suppression of IL-8 production by monocytes, keratinocytes, or fibroblasts (3), such inhibition in hOCL was only weakly dose dependent and did not surpass a 30% decline in stimulated IL-8 levels even at hormonal concentrations as high as 10-7 M, in contrast to the potent inhibitory effects of dexamethasone. Finally, neither of the bone resorption inhibitory hormones, calcitonin or 17ß-estradiol, directly influenced either basal or IL-1{alpha}-stimulated IL-8 release from hOCL, although hOCL expressed mRNA for each receptor and have responded in other ways to calcitonin (46) and 17ß-estradiol (47). Estrogen also does not affect basal or stimulated IL-8 release by hBMS, hOB, or human MG-63 osteosarcoma cells (19, 48, 49), although estrogen can inhibit cytokine-induced MCP-1 production in fibroblasts (50). Thus, hOCL production of IL-8 is predominantly regulated by inflammatory mediators and not by other osteotropic factors or hormones. Recent work has also suggested more complex temporal modes of regulating IL-8 production in hOCL, such as via estrogen modulation of IL-1 receptor expression in hOCL, which subsequently modifies IL-1-mediated induction of IL-8 mRNA (47).

Evidence for the in vivo expression of IL-8 by resorbing hOC and its regulation by inflammatory stimuli was obtained from in situ hybridization studies of human bone tissues. Thus, IL-8 mRNA levels were higher in the resorbing hOC of rheumatoid arthritic pannus than in hOC of giant cell tumors of bone. Although both of these conditions represent pathological states in which OC numbers are dramatically increased over the low number usually observed in human bone, inflammation is not considered a hallmark of giant cell tumors of bone, whereas rheumatoid arthritis is an autoimmune disease characterized by chronic joint inflammation that leads to a progressive destruction of cartilage and bone tissue in the afflicted joint (30, 51, 52, 53). Preliminary in situ hybridization studies on human osteonecrotic bone samples have similarly evidenced only low IL-8 mRNA levels in the resorbing hOC of this noninflammatory tissue (15). Therefore, hOC recruited into the developing pannus region in rheumatoid arthritis may respond to local stimulatory signals elaborated in this microenvironment by increasing their synthesis of IL-8, which could then contribute to the maintenance and amplification of the inflammatory response in this disorder. Chemokines, including IL-8, play key roles in both the initiation and maintenance phases of the evolutionary pathogenesis of rheumatoid arthritis by virtue of their capacity to recruit and activate leukocyte populations (monocytes and neutrophils) infiltrating the synovial space and joint tissue, to stimulate synovial cell proliferation and pannus formation at the juncture of the synovium lining the joint capsule with the cartilage and bone, and to activate endothelial cells and promote neovascularization, which further favors the delivery of immune cells from the vascular compartment into the inflamed joint (51, 52, 53, 54). Pre-OC are also likely to be recruited, whereupon their exposure to locally high concentrations of inflammatory mediators may promote their development and bone-resorptive activity. Whereas limiting chemokine levels appear optimal for the recruitment and passage of blood cells across the vascular endothelium into the inflamed tissue site, adhesion and activation of cells once in this location typically require high saturating concentrations of chemokines (55), such as those detected (up to 220 ng/ml IL-8) in the synovial fluid and serum of rheumatoid arthritic individuals (38) and shown here to be released by cultured hOC and hOCL. Consequently, hOC may significantly contribute to such IL-8 increases and thereby exert paracrine inflammatory or immunoregulatory influences on other cells infiltrating or present within the arthritic lesion.

Both stimulatory and suppressive factors are commonly coproduced during inflammation, and their relative balance is thought to provide a mechanism for the sensitive and rapid modulation of the inflammatory response (38). Although IL-8 appears to inhibit overall bone resorption by isolated avian (17) or rat (18) OC, this has been attributed at least in the rat system to a decrease in the proportion of OC actively resorbing bone due to an IL-8-stimulated increase in their motility, rather than to a suppression of the actual bone-resorptive capacity of individual OC. Therefore, it is also possible that hOC-derived IL-8 exerts important autocrine actions, similar to those seen in neutrophils (30), which might enhance OC degradation of bone at inflammatory sites. This is consistent with the elevated numbers of OC and augmented bone resorption activity exhibited in rheumatoid arthritis seen here and in other studies. One potential resorption-stimulating mechanism might be via IL-8 triggering of OC release of superoxides (56), reactive oxygen species involved in OC bone resorption (57, 58). In addition, hOC-derived IL-8 might elicit recruitment and activation of additional OC, because select chemokines, including IL-8 (17, 18) and MIP-1{alpha} (59, 60), promote migration or chemotaxis of avian, rat, or human OC (42, 47, 48, 59, 61, 62). In conclusion, we have shown that isolated bone-resorptive hOC and in vitro formed hOCL synthesize and secrete high levels of the potent proinflammatory chemokine IL-8, that mRNA and cytokine levels for IL-8 are further raised in hOCL in response to various inflammatory stimuli, and that IL-8 mRNA expression is localized in situ to resorbing hOC of human bone tissues, wherein it is particularly evident in inflammatory rheumatoid arthritic pannus. Therefore, hOC-derived IL-8 may serve as an important autocrine/paracrine mediator of bone cell physiology and immunoregulation involved in normal or pathological bone remodeling.


   Acknowledgments
 
The authors are indebted to Drs. Curt Merkel and William Maloney for supplying us with femoral head segments of human bone derived from osteoporotic patients from which we could isolate numerous human bone-resorptive osteoclasts. We also greatly appreciate the assistance of Mr. Fred Anderson and Ms. Sheng Zhang in the preparation of human osteoclasts and bone marrow-derived osteoclast-like cells; Mr. Leonard Rifas, Ms. Aurora Fausto, and Ms. Linda Halstead in the preparation of human bone marrow-derived stromal cells and osteoblast-like cells; and Dr. Oscar Joost, Dr. Alan Dean, and Mr. Ivan Zeiss in performing the Northern blot analysis of IL-8 mRNA steady state expression.


   Footnotes
 
1 This work was supported by NIH Grants AR-32087 and DE-06891 (to P.O.) and DK-46773 (to S.G.), and a Mineral Metabolism Fellowship (to Y.C.). Back

Received January 27, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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