Bone Explants Is Associated with Suppression of Osteoclastic Bone Resorption

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1 Indution ofnitri Oxide in Osteoblasts Can Regulate Osteolasti Resorption 1465 Induible Prodution of Nitri Oxide in Osteoblast-like Cells and in Fetal Mouse Bone xplants Is Assoiated with Suppression of Osteolasti Bone Resorption Clemens W. G. M. L6wik, Peter H. Nibbering,* Marjan van de Ruit, and Sorates. Papapoulos Departments ofndorinology and *Infetious Diseases, University Hospital Leiden, Rijnsburgerweg 1, 2333 AA Leiden, The Netherlands Abstrat Nitri oxide (NO) has been suggested to be involved in the regulation of osteolast ativity. Sine osteoblasts, through the release of various fators, are the main regulators of osteolasti resorption, first we have investigated whether osteoblastlike ells and fetal mouse long bone explants are able to produe NO. Seond, we have assessed the effet of NO on osteolasti resorption in whole bone ultures. In this study we show that primary rat osteoblast-like ells as well as the lonal rat osteoblast-like ell line UMR-16, stimulated with IFN-'y together with TNF-a and LPS, produe NO, measured as nitrite prodution. IL-la enhaned while TGF-#2 inhibited TNF-a + IFN-y + LPS-stimulated NO prodution in UMR-16 ells dose dependently. Both the ytokines, however, had no effet when given alone. The ompetitive inhibitor of NO prodution, NG-monomethyl-arginine (L-NMMA), and yloheximide abolished the inrease in nitrite prodution indued by TNF-a + IFN-'y + LPS, while hydroortisone had no effet, as previously reported for hondroytes. Caliotropi hormones had either no effet 11,25(OH)2D31 or had a small inhibitory effet (parathyroid hormone) on stimulated NO prodution. Furthermore, we found that in ultured fetal mouse long bone explants the ombination of TNF-a + IFN-'y + LPS as well as the NO donor sodium nitroprusside ould inhibit osteolasti resorption, measured as 45Ca release. The inhibition of resorption was prevented by onurrent administration ofl-nmma. Histologial evaluation revealed that the TNF-a + IFN-'y + LPS-indued inhibition of 45Ca release was assoiated with a derease in the number of tartrate-resistant aid phosphatase-positive osteolasts. We propose that the NO prodution by osteogeni ells (osteoblasts and hondroytes) may represent an important regulatory mehanism of osteolasti ativity espeially under pathologial onditions harateried by release of boneresorbing inflammatory ytokines. (J. Clin. Invest : ) Key words: nitri oxide * osteoblasts * bone - osteolasts * resorption Parts of the findings desribed in this paper have been published in abstrat form ( Calif Tissue Int. 52 [Suppl. 1] :S13. [Abstr. 49]). Address orrespondene to Dr. C. W. G. M. Lfwik, Department of ndorinology, University Hospital, Bldg. 1, C4-R89, Rijnsburgerweg 1, 2333 AA Leiden, The Netherlands. Reeivedfor publiation 2 August 1993 and in revisedform I November J. Clin. Invest. The Amerian Soiety for Clinial Investigation, In /94/4/1465/8 $2. Volume 93, April 1994, Introdution Nitri oxide (NO)' is a reently identified messenger moleule regulating a wide range of funtions throughout the body (for review see referenes 1-3). NO is synthesied from L-arginine by NO-synthase (NOS), and an at as a neurotransmitter in the brain and peripheral nervous system; it has been identified as the endothelial-derived relaxation fator, whih binds to guanylate ylase leading to GMP formation and subsequent smooth musle relaxation. NO an also ontribute to immune funtion sine NO produed by marophages is involved in their tumoriidal and bateriidal ations (4-6). At present three distint forms ofnos have been identified and loned (7-9). In neurons and brain (erebellar type) and blood vessels (endothelial type), NOS is onstitutive and Ca2" dependent (7, 8), whereas in marophages (9), hepatoytes (1), fibroblasts (11), and hrondroytes (12-14) (marophage type), the enyme ativity is indued by ytokines and endotoxins. Using isolated osteolasts, it has reently been shown that NO ats diretly on the osteolast to produe a major shape hange. These shape hanges were assoiated with a redution of bone resorption after a 24-h inubation of isolated osteolasts on devitalied bone slies. These effets of NO were not mediated by GMP or hanges in intraellular alium ( 15 ). These authors suggested that NO-produing endothelial ells in bone marrow in the proximity of osteolasts ould regulate osteolast ativity. However, it is well established that the osteogeni ells (osteoblasts and hondroytes) are the main regulators of osteolasti resorption, through khe release of proteases, ytokines, and other fators (16, 17). The aim of this study was to investigate first whether osteoblast-like ells and fetal mouse long bones are able to produe NO, and seond, to assess the effet ofno on osteolasti resorption in fetal mouse long bone ultures. Methods Materials. amm and RPMI were purhased from Gibo Laboratories (Grand Island, NY), and peniillin, streptomyin, and FCS were from Flow Laboratories (Amstelstad, Zwanenburg, The Netherlands). Reombinant human TNF-a was from Boehringer Ingelheim (Ingelheim, Germany). Reombinant human IL-la was a gift from Dr. P. Lomedio (Hoffman-La Rohe In., Nutley, NJ), reombinant rat IFN-'y from Dr. P. H. van der Meide (Institute of Applied Radiobiology and Immunology, Rijswijk, The Netherlands), and reombinant TGF-32 from Dr. J. H. M. Feijen (Sando, Basel, Switerland). LPS from sherihia oli was obtained from Difo Laboratories In. (Detroit, MI), and NG-monomethyl-L-arginine (L-NMMA) and sodium 1. Abbreviations used in this paper: L-NMMA, NG-monomethyl-L-arginine; NO, nitri oxide; NOS, nitri oxide synthase; N2-, nitrite; PTH, parathyroid hormone.

2 nitroprusside (SNP) from Calbiohem-Behring Corp. (San Diego, CA). All other hemials used were of highest quality available. Cell ultures. Primary ultures of rat osteoblast-like ells were prepared from 2-d-old fetal rat alvaria by the sequential ollagenase method ( 18). ROB were ultured in a 24-well ulture luster (Greiner Labortehnik, Frikenhausen, Germany) in amm supplemented with 1% FCS, gentamyin (9 mg/ 1 ml), and gluose ( 1 mg/ 1 ml). UMR-16, a lonal rat osteogeni saroma ell line with a harateried osteoblast phenotype ( 19), was a gift of Dr. T. J. Martin (St. Vinent's Institute for Medial Researh, Melbourne, Australia). UMR-16 ells were ultured in amm supplemented with 5% FCS and antibiotis. Bone resorption assay. Pregnant Swiss albino mie were injeted intraperitoneally with 3 MCi 45Ca (sp at, 1 Ci/mmol; Amersham International, Amersham, UK) on day 16 of gestation, whereby vaginal plug disovery was defined as day of gestation. 1 d later the animals were killed by servial disloation. The 45Ca-prelabeled bone shafts ofradii were diseted out in Hepes-buffered Hank's solution (ph 7.4) as desribed (2). Shafts of radii were ultured in a 24-well plates in amm + 1% heat-inativated FCS and antibiotis in a final volume of 5,ul. xplants were preultured for 24 h to allow the alium exhange with the medium to reah a steady state and then ultured for 4 d in similar medium with or without the agents to be tested. Cultures were performed at 37 C in a humidified atmosphere of5% CO2. At the end ofthe ulture period, 25 Ml ofthe medium was used for determination ofthe amount of45ca released from the bone explants, whereas the other 25 ;d was used for determination ofthe nitrite ontent. Residual alium was extrated from the bones in.5 ml 5% TCA for 24 h. In some experiments bones were fixed and proessed for histology. The amounts of 45Ca in the ulture media and in the dealifiation fluids were determined in a liquid sintillation ounter. Calulation ofosteolasti resorption. Resorption was expressed as the perentage 4uCa in the prelabeled explant that is released during inubation (% 45Ca release). The values were orreted for physiohemial alium exhange by substration of the perentage 45Ca release from a dead bone (perentage killed ontrol [% KCo]). Bones were killed by three yles of freee-thawing. Osteolasti resorption is thus alulated as: x 1% - % KCo release. 45Ca release into medium (pm) total uca inorporated in bone (pm) Histology. At the end of the ulture period, the bone explants were fixed in 1% neutral-buffered formalin for 24 h at 4 C and subsequently dealified in 5% formi aid and 5% formalin for 3 h at 4 C. The explants were then proessed for demonstration of tartrate-resistant aid phosphatase ativity aording to the en blo staining method of Sheven et al. (21), using naphtol AS-BI phosphate as substrate, pararosalin as oupler, and 1 mm L-tartrate as inhibitor. Paraffin setions 5 Mm thik were ounterstained with hematoxylin. Nitrite prodution. NO was measured as nitrite prodution in ulture medium of onfluent ultures after 24 h of inubation. Nitrite is the stable end produt of NO and has been shown to be a good refletion of NO prodution. The amount of nitrite (NO2-) released by ells or bone explants was determined using the Griess reagent onsisting of 1% sulfanylamide,.1% naphtylethelene-diamine-dihydrohloride, and 2.5% H3PO4. Briefly, 75 Ml ulture supernatant was mixed with 75 Ml Griess reagent and inubated in a 96-well plate for 15 min at room temperature under ontinuous shaking. N2- onentration, proportional to OD5, was determined using a mirotiterplate reader (Thermomax'T, Moleular Devies, Menlo Park, CA) with referene to a standard urve of serial dilutions of NaNO2. Total ellular protein ontent was measured with the BCA protein assay (Piere Chemial Co., Rokford, IL). The results are expressed as nanomoles NO2- per miligram ellular protein. Statistis. Values are expressed as means±sm. Statistial differenes between the values were examined by one-way ANOVA for multiple omparisons followed by Fisher's test. Results NO prodution by rat osteoblast-like ells. In unstimulated onfluent ultures of primary fetal rat osteoblast-like ells a spontaneous prodution of NO2- was found that was stimulated signifiantly after a 24-h inubation with IFN-y (1 U/ ml) and LPS (1 ng/ml) or a ombination of the two. TNF-a (1 ng/ml) alone did not stimulate N2- prodution, but in ombination with IFN-'y or LPS enhaned NO2- prodution signifiantly ompared with the single stimuli. The most potent stimulus of NO2- prodution was the ombination of TNF-a + IFN--y + LPS (Fig. 1 A). Similar results were found when primary mouse osteoblasts were used (results not shown). We also performed the same experiments with the lonal rat osteogeni saroma ell line UMR- 16, whih has a well-harateried osteoblast-like phenotype (19). UMR-16 ells did not produe N2- spontaneously, but when the ells were stimulated by TNF-a together with LPS or IFN-'y, or the ombination ofthe three, a signifiant inrease in NO2- prodution was measured (Fig. 1 B). Sine the UMR- 16 ells are osteoblastlike ells of lonal origin and an be stimulated to produe NO2 similar to ROB, they were used in all further studies unless otherwise indiated. -5 L.. 15 on C 1-5 * * r_-i * C * L ~+.5 al -JL LL a Z Z Z a- _ 2. C) U 21- an.-.-.) -l U) -j In a LL L I- ffi ~ ~ + >- + h + Z + ~~~~~~~~~~~~~~~~~~n -~~~~~~ Figure 1. ffet of LPS (1 ng/ ml), IFN-'y (1 U/ml), TNF-a (1 ng/ml), or ombination of these on NO2- prodution by (A) primary rat osteoblast-like ells (ROB), and (B) the lonal rat osteoblast-like ell line UMR-16. Confluent ultures were inubated with the additives for 24 h. Data (nmol NO2-/mg ellular protein) are means±sm of three experiments, eah performed in tripliate. *P <.5, **P <.1, ***P <.1. NS, not signifiant Liwik et al

3 Indution ofnitri Oxide in Osteoblasts Can Regulate Osteolasti Resorption 1467 Time ourse of NO prodution. To determine the time ourse of the response, UMR-16 ells were ultured to onfluene and stimulated with TNF-a + IFN-y + LPS. Signifiant amounts of N2- were measured after a lag period of 6 h, and inreased further during the next 18 h followed by a muh slower seondary inrease up to 72 h (Fig. 2). In all further experiments N2- prodution by ells was determined in the ulture medium after 24 h of inubation with or without stimuli. Dose-response studies with a ombination oftnf-a + LPS + IFN-,y. Three dose-response experiments with a ombination of TNF-a + LPS + IFN--y were performed. In these the onentration of one of the stimuli varied while the onentrations ofthe other two were kept onstant. All stimuli inreased NO prodution dose dependently (Fig. 3). Partial harateriation ofno indution. The inrease in the amount of NO2- indued by TNF-a + LPS + IFN-y ould be inhibited by simultaneous addition of I mm ofthe ompetitive inhibitor of NOS, L-NMMA, or by 1-6 M yloheximide (Table I). Hydroortisone, at onentrations of 1-8 and I-7 M, had no signifiant effet on the TNF-a + LPS + IFN-ystimulated NO2- prodution. ffets of IL-ila parathyroid hormone (PTH), and dibutyryl-amp on stimulated NO prodution. IL- 1 a synergistially stimulated TNF-a + IFN-,y or TNF-a + INF-y + LPS-indued N2- prodution, but was ineffetive on its own (Fig. 4) M PTH( 1-34) alone or in ombination with TNF-a + IFN-y did not affet N2- prodution in UMR-16 ells. However, in the presene of the ombination of TNF-a + IFN-,y + LPS, PTH signifiantly inhibited N2- prodution. The inhibitory effet ofpth ould be mimiked by. 1 and 1. mm dbamp (Fig. 4). Dose-response of IL-Ia and TGF-,32 on stimulated NO prodution. Fig. 5 shows the effet of various doses of IL-la and TGF-32 on TNF-a + IFN-,y + LPS-stimulated NO prodution. IL- 1 further inreased stimulated NO prodution dose dependently. In ontrast, TGF-32 dose-dependently inhibited TNF-a + IFN-y + LPS-indued NO prodution. TGF-,32 also inhibited TNF-a + IFN--y-stimulated NO prodution but was ineffetive on its own (results not shown). '. Q 3 C'D C 3 to * 2I o, UMR-16 Ho A ^ ^ 2 4 s s time (h) Figure 2. Time ourse of N2- prodution by onfluent ultures of UMR- 16 ells stimulated with a ombination of TNF-a (1 ng/ml) + IFN-y ( 1 U/ml) + LPS ( 1 ng/ml). Data (nmol N2-/mg ellular protein) are the means of two experiments, eah performed in quadrupliate. Variation in the mean values are never > 5%. (a 2 - LPS IFN-y i Tu 3 ) C (a 3 C 2(. 1-2C 11 1 ng/ml :1U/ml rhtnf-a (ng/ml) TNF-a :1nnml IFN- Y : 1 U/ml. TNF- a + IFNy I. 1- UMR LPS (ng/ml) - - LPS : 1 ngfml TNF-a :1ngtml UMI-1 U- I ~~~~~ LPS + TNF- a rrifn-- (U/ml) Figure 3. Dose-response urves of the effets of ombinations of TNF-a + LPS + IFN--y on N2- prodution by UMR-16 ells after 24 h of inubation. In these experiments the onentrations of two of the stimuli were kept onstant whereas the onentration of one of the stimuli varied. (A) Dose-response of reombinant human TNF-a in the presene of 1 ng/ml LPS and 1 U/ml IFN-y. (B) Dose-response of LPS in the presene of 1 ng/ml TNF-a and 1 U/ml IFN--y. (C) Dose-response of reombinant rat IFN-y in the presene of 1 ng/ml LPS and 1 ng/ml TNF-a. Data (nmol NO2-/mg ellular protein) are the means of two experiments, eah performed in quadrupliate. Variation in the mean values are never > 5%. Relation between NO prodution and osteolasti resorption in ultured fetal mouse long bones. We next investigated whether NO prodution ould be indued in fetal mouse long bone explants and whether this is related to hanges in osteo-

4 .~~ Table I. ffets of Various Compounds on N2- Prodution by the Osteoblast-like Cell Line UMR-16 NO2/ellular Additives protein nmol/mg None ND L-NMMA ND TNF-a + IFN-,y 1.65±.2 TNF-a + IFN-y + L-NMMA ND TNF-a + IFN-'y + LPS 26.5±1.1 TNF-a + IFN-'y + LPS + L-NMMA 5.25±.4 TNF-a + IFN-,y + LPS + HC (1-8 M) 25.±1.2 TNF-a + IFN-y + LPS + HC (I-' M) 24.±1.5 TNF-a + IFN-y + LPS + CHX 4.±.5 Confluent ultures of UMR- 16 ells were treated with various additives for 24 h. Conentrations used were 1 ng/ml TNF-a; 1 U/ml IFN-y; 1 ng/ml LPS; 1 mm L-NMMA; 1-' and 1-7 M hydroortisone (HC); 1-6 M yloheximide (CHX). Data, expressed as nmol NO2-/mg ellular protein, are means±sm of three experiments, eah performed in tripliate. ND, not detetable. lasti resorption. TNF-a (1 ng/ml) + IFN-'y (1 U/ml) + LPS ( 1 ng/mnl) indued a signifiant inrease in the amount of NO2- in the ulture supernatant ofthese bone explants after 4 d of ulture (Fig. 6 A). This inrease in NO prodution was assoiated with a signifiant inhibition of resorption measured as 45Ca release (Fig. 6 B). Addition of 1 mm L-NMMA signifiantly inhibited TNF-a + IFN--y + LPS-stimulated NO2- prodution from 4.±.2 to.9±.2 nmol NO2-/well (P <.5), and ould prevent the suppression of osteolasti resorption (Fig. 6). Control resorption, however, was not influened by nmol NO2 -/mg ellular prot ] 4.1 CY) ẕa 4 - UMR m o a U LIS + TNF- a+ IFN- y o rhil-1 a (ng/ml) LUMR-1l6-. TNF- a + IFN- y + LPS - ~~~~~~~~~~~~~~~~~~~~\~~~~~~~~~~~~~~~~~~~ rhtgfp 2 (ng/mi) Figure 5. The effet of various doses of (A) reombinant human ILla and (B) reombinant human TGF-#2 on LPS (1 ng/ml) + TNF-a (1 ng/ml) + IFN-'y (1 U/ml)-stimulated N2 prodution by onfluent ultures of UMR-16 ells after 24 h of inubation. Data (nmol NO2-/mg ellular protein) are the mean of two experiments, eah performed in quadrupliate. Variation in the mean values was never > 5%. 1 ontrol IL-1 (x PTH TNF- (L+IFN-y TNF- ( +IFN-Y +IL-1 TNF-(t +IFN-y+PTH TNF-(x +IFNy+LPS TNF-(x +IFN-y+LPS+IL-1 a TNF-x +IFNY+LPS+PTH TNF- +IFN-Y + LPS +dbamp.1 TNF- +IFNi + LPS +dbamp 1. I NS I X///////X//IM//X x~~~~~~..~~~ _ ~~x. _ ~x UMR Figure 4. ffet of 4 nm PTH (gray bars), 1 ng/ml IL- la (hathed bars), and.1 and 1. mm dbamp (blak bars) on basal or stimulated N2- prodution by onfluent ultures of UMR- 16 ells. The ells were either unstimulated or stimulated for 24 h with TNF-a ( 1 ng/ml) + IFN-'y ( 1 U/ml) or with TNF-a + IFN-'y + LPS (1 ng/ml). Data (nmol NO2-/mg ellular protein) are means±sm ofthree experiments, eah performed in tripliate. ***Signifiantly different from TNF-a + IFN-'y; P <.1. NS, not signifiant. xsignifiantly different from TNF-a + IFN-'y + LPS; P <.5, 'P <.1. L-NMMA. These findings were onfirmed by histologial evaluation of the long bones (Fig. 7). In the presene of TNF-a + IFN-'y + LPS (Fig. 7 C), almost no osteolasts were found but simultaneous addition of L-NMMA (Fig. 7 d) totally restored osteolasti resorption to ontrol level (Fig. 7 b). In Fig. 8 A, it is shown that the NO-releasing agent sodium nitroprusside stimulated the N2- prodution in the bone ultures dose dependently, both in the absene and presene ofpth ( M). Furthermore, sodium nitroprusside inhibited basal- as well as PTH-indued osteolasti resorption measured as net 45Ca release in a dose-dependent fashion. Disussion This study shows that various ytokines or ombinations of these with LPS an indue NO prodution in primary rat osteoblast-like ells as well as in the lonal rat osteoblast-like ell line UMR- 16. Spontaneous prodution of NO was, however, observed only in ultures of primary osteoblast-like ells. Whether this was due to other ells ontaminating the primary osteoblast ultures (e.g., endothelial ells) or to the presene of 1468 Lowik et al.

5 Indution ofnitri Oxide in Osteoblasts Can Regulate Osteolasti Resorption Cy 2 *1I D i a C ' A ontrol T orol 7- L-NMMA T T TNF+IFN+LPS L-NMMA T+I+L+L-NMMA Figure 6. ffet of LPS (1 ng/ml) + IFN-y (1 U/ml) + TNF-a (1 ng/ml) in the absene or presene of 1 mm L-NMMA on (A) N2- prodution, and (B) net %45Ca release by fetal mouse long bones (shafts of 17-d-old radii) after 4 d of ulture. Data (nmol NO2-/well or net %45Ca release) are means±sm of four ultures. *Signifiantly different from ontrol, P <.5. Compared with LPS + IFN-y + TNF-a treated, P <.5. a onstitutive NOS in normal osteoblast-like ells (as opposed to those of malignant origin) needs to be addressed in further studies. Similar to other ell types ontaining an induible NOS, the inrease in NO prodution in the UMR-16 ells started after a lag period of 6 h and ontinued thereafter. We demonstrated that the ompetitive inhibitor of NO prodution, L-NMMA, bloked stimulated NOS ativity and that the indution of NO prodution ould be inhibited by yloheximide. Similar to hondroytes (12-14), but in ontrast to other ell types ontaining an induible NOS (22, 23), the NO2- prodution ould not be inhibited by hydroortisone. In ontrast to hondroytes ( 12-14), but similar to fibroblasts ( 11 ), IL- 1 a alone ould not indue N2- prodution in UMR- 16 ells but stimulated NO2- prodution synergistially when ombined with TNF-a + IFN-y or TNF-a + IFN-y + LPS. TGF-f32 alone had also no effet on NO2- prodution but ould dose dependently inhibit TNF-a + IFN-y or TNF-a + IFN-,y + LPS-indued NO2- prodution. This is in line with findings in marophages in whih TGF-#31, 2, and 3 are all able to inhibit indution of NOS ativity (24). The major bone resorbing hormone, PTH, had no effet on NO2- prodution when added alone or in ombination with TNF-a + IFN-,y, but had a small signifiant inhibitory effet on TNF-a + IFN-y + LPS-indued N2- prodution. This was probably due to its well-known stimulatory effet on intraellular AMP sine the inhibition ould be mimiked by the AMP analogue, dibutyryl AMP. The ative metabolite of vitamin D3, 1,25 (OH)2D3, also a stimulator of bone resorption, had no effet on basal as well as stimulated NO prodution (results not shown). Apart from mouse and rat osteoblast-like ells, NO prodution ould also be indued in fetal mouse long bone explants by TNF-a + IFN-y + LPS and by the NO-releasing ompound sodium nitroprusside. The indution of NO prodution by TNFa + IFN-y + LPS and by sodium nitroprusside at onentrations >.1 mm was assoiated with a signifiant inhibition of basal osteolasti resorption. The latter is in line with the findings of MaIntyre et al. (15), who reported that NO indued a rapid shape hange in isolated osteolasts that was assoiated with an inhibition of bone resorption when the osteolasts were inubated for 24 h on devitalied bone slies. In unstimulated fetal mouse bone shafts, ontaining mainly osteoblasts, hondroytes, fibroblasts, and osteolasts, there was no detetable NO prodution and 45Ca release ould not be inhibited by L-NMMA, indiating that basal osteolasti resorption is not regulated by a onstitutive NO prodution. The inhibition of bone resorption by TNFa + IFNy + LPS, however, ould be prevented by onurrent addition of L-NMMA, indiating that this inhibition was NO mediated, but in the presene of L-NMMA resorption was not inreased above ontrol levels. The reason for this might be that IFN-y, apart from an effet through NO, may also at through other pathways affeting osteolasti resorption, i.e., inhibition of osteolast formation, as has already been suggested by Gowen et al. (25). Histologial evaluation revealed that the LPS + IFN-y + TNF-aindued inhibition of 45Ca release was assoiated with a derease in the number of tartrate-resistant aid phosphatasepositive osteolasts. The absene of any evidene of ell or tissue damage makes a possible toxi effet of NO on these ells highly unlikely. The exat level of ation of NO an not be dedued from these experiments. To determine if the NO-mediated derease in osteolasts is due to a derease in osteolast formation or just to a diret inhibition of osteolast ativity as desribed by MaIntyre et al. (15), further studies are warranted. It is remarkable that only NO2- levels > 2. nmol/ well are related to inhibition of basal bone resorption sine in the presene of.1 mm sodium nitroprusside or TNF-a + IFN-y + LPS + L-NMMA, the NO2- levels were still signifiantly elevated (2.±.5 and.9±.2, respetively) ompared with ontrol, but resorption was unaffeted. However, PTHstimulated bone resorption was already signifiantly inhibited by.1 mm sodium nitroprusside. Although this might just be due to the sensitivity of our resorption assay, it is possible that stimulated resorption is muh more sensitive to NO-mediated inhibition than basal resorption. In this way basal resorption is not immediately influened by small hanges in NO onentrations onstitutively produed by other ells, like endothelial ells, neutrophils, mast ells, platelets, and neurons, present in or near the bone ompartment. However, it is likely that vasoative, neuronal ative, and other rapidly ating ativators of the onstitutive NOs ativity an affet bone resorption through an NO-mediated pathway.

6 ta b d ViZ~ ~ ~ ~ ~ ~ ~ ~ ~ _ W~ Figure 7. Transversal setions through shafts of 17-d-old radii after 4 d of ulture. The bone explants are stained for tartrate-resistant aid phospatase (TRAP) ativity and setions are ounter stained with hemotoxylin (as desribed in Methods). Representative setions are shown of (a) "killed" ontrol (three yles of fr-eee-thawing), (b) ontrol, () LPS (1 ng/ml) + IFN-'y (1 U/mi) + TNF-a (1 ng/ml)-treated explants, and (d) LPS + IFN-,y + TNF-a-treated explants in the presene of 1 mm L-NMMA. In the "killed" ontrol (a) there is no sign of ellular ativity. In the ontrol ultures (b) TRAP-positive osteolasts, are found espeially at the sites were trabeulae are present (arrows). In the presene of LPS + IFN-'y + TNF-a () almost no TRAP-positive osteolasts are found. Simultaneous addition of 1 mm L-NMMA (d) totally restores LPS + IFN-'y + TNF-a-inhibited osteolasti resorption bak to ontrol levels. Note the reourene of TRAP osteolasts (arrowse 147 Lowik et al

7 a a1) ('3 Cu a, a, a, ontrol sodium nitroprusside (mm) 1 * PTH sodium nitroprusside (mm) ontrolo PTH sodium nitroprusside sodium nitroprusside (mm) T (mm) Figure 8. The effet of the NO donor sodium nitroprusside in the absene or presene of 4 nm PTH on (A) NO- prodution, and (B) net %45Ca release by fetal mouse long bones (shafts of 17-d-old radii) after 4 d of ulture. Data (nmol NOR/well or net %45Ca release) are means±sm of four ultures. *Signifiantly different from ontrol; P <.5, **P <.1, ***P <.1. xcompared with PTH treated; P <.5, 'P <.1, 'P <.1. An important question that arises whenever animal tissues or ells are used is whether the findings an be diretly appliable to human physiology. Although indution of NO synthesis by ytokines has not been found in human marophages, it was reently shown that human artiular hondroytes express an induible NOS ( 14). Compared with the NOS present in rabbit hondroytes ( 13), the human enyme differs in its sensitivity to alium and gluoortioids. However, it was shown that the NOS in both speies belonged to the same family as the NOS expressed in rodent marophages ( 14). As hondroytes are osteogeni ells, these findings in rodent bone ells are likely to be relevant to human ells as well. At this stage we an only speulate about the linial relevane of our observations. Our hypothesis is that NO prodution may represent a protetive mehanism against unopposed bone resorption during pathologial onditions, suh as for example during inflammation. As the inflammatory ytokines IL-I and TNF as well as LPS are known stimulators of osteolasti bone resorption (26), one would expet onsiderable bone loss duringthe ourse ofinflammatory proesses, whih is not the ase. IFN-y, whih is also produed during inflammation by ativated T lymphoytes, appears to be the key regulator of this protetive mehanism. In the presene of TNF-a, IL- 1, and LPS, IFN-'y indues NO prodution, whih enhanes the immune funtion but in addition prevents bone resorption indued by IL- 1 and TNF. If this hypothesis is orret then in onditions with impaired IFN-y prodution one would expet to find inreased bone loss. This is in fat the ase in rheumatoid arthritis in whih ativated T lymphoytes have been shown to have an impaired prodution of IFN-y ompared with ontrols (27). The systemi stimulators of bone resorption, PTH and 1,25(OH)2D3, involved in the normal ontrol of bone metabolism have no effet on NO prodution on their own or in the presene of IFN-y. This strengthens further the hypothesis that the stimulation of the NO pathway appears to be an important regulator of osteolast ativity only under pathologial onditions harateried by release of bone resorbing inflammatory ytokines. Our hypothesis is supported by the findings of Gowen et al. (25), showing that IFN-,y strongly inhibited bone resorption indued by TNF or IL-l but not that indued by PTH and 1,25(OH)2D3. In onlusion, NO produed by osteogeni ells (osteoblasts and hondroytes) may be an important regulator of osteolast ativity espeially in onditions harateried by elevated loal release of inflammatory, bone resorbing ytokines. These findings might open new perspetives in the approah of bone disorders harateried by inreased osteolasti bone resorption like high turnover osteoporosis, hyperalemia due to metastati bone disease, Paget's disease, and rheumatoid arthritis. Aknowledgments This work was partially supported by the Netherlands Organisation for the Advanement of Pure Researh (NWO, ). Referenes 1. Monada, S., R. M. J. Palmer, and. A. Higgs Nitri oxide: physiology, pathophysiology and pharmaology. Pharmaol. Rev. 43: Nathan, C Nitri oxide as a seretory produt of mammalian ells. FASB (Fed. Am. So. xp. Biol.) J. 6: Lowenstein, C. J., and S. H. 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