with Tyrode's solution, and cultured in 9 cm 2 tissue culture plates containing ol-MEM with penicillin (33/~g/ml), streptomycin (50/~g/ml) and 10% fetal calf.
Bioscience Reports, Vol. 10, No. 1, 1990
Bradykinin Stimulates Prostaglandin Formation in Isolated Human Osteoblast-Like Cells
E2
Osten Ljunggren, ~'3 Jan Rosenquist, 2 Maria Ransj6 ~ and U l f H. Lerner ~ Received November 20, 1989 The effect of bradykinin on prostaglandin E 2 formation in cells from human trabecular bone has been studied. The cells responded to parathyroid hormone with enhanced cyclic AMP formation and were growing as cuboidal-shaped, osteoblast-like cells. In these isolated human osteoblast-like cells, bradykinin (1 #tool/l) caused a rapid (5 min) stimulation of prostaglandin E2 formation. This finding indicates that human osteoblasts are equipped with receptors for bradykinin linked to an increase in prostaglandin formation. KEY WORDS: Bradykinin; prostaglandin E2; osteoblasts.
INTRODUCTION
We have previously reported that the inflammatory mediator bradykinin enhances bone resorption in vitro (1, 2). The mechanism of action is dependent on a bradykinin induced increase of endogenous prostaglandin formation in bone tissue, with subsequent stimulation of bone resorption. Evidence for this view include the findings that prostaglandins stimulate bone resorption in vitro (3), bradykinin induced bone resorption in vitro is blocked by agents that inhibit prostaglandin synthesis e.g. indomethacin and hydrocortisone (2), bradykinin stimulates the formation of prostaglandin E2 (PGE2) and prostacyclin in neonatal mouse calvarial bones (4) and finally, bradykinin stimulates PGE2 and prostacyclin formation in isolated murine calvarial osteoblast-like cells and in a cloned, murine, osteoblastic cell lineage (MC3T3-EI; 4) as well as in isolated rat eosteoblast-like cells (5). In view of these findings, we have investigated the effect of bradykinin on 1 Department of Oral Pathology, University of Umefi, S-901 87 Umefi, Sweden. 2 Department of Oral Surgery, University of Umefi, S-901 87 Umefi, Sweden. 3 To whom correspondence should be addressed. 121 0144-8463/90/0200-012150600/0~) 1990PlenumPubhshmgCorporation
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human osteoblasts in order to evaluate whether bradykinin may be a mediator of inflammatory induced bone resorption in human diseases such as periodontitis, rheumatoid arthritis or osteomyelitis. MATERIALS AND METHODS Materials Bradykinin and des-arg9-bradykinin were from Sigma Chemical Co., St. Louis, MO; USA; alpha modification of Minimum Essential Medium (ol-MEM) and fetal calf serum from Flow Laboratories, Irvine, Scotland; synthetic bovine parathyroid hormone (PTH 1-34) with a potency of 6800 IU/mg from Bachem, Switzerland; the radioimmunoassay (RIA) kits for PGE2 and cyclic AMP from' New England Nuclear Chemicals, Dreieich, FRG; multiwell plastic culture dishes from Costar, Cambridge, MA, USA. Isolation of Human Osteoblast-Like Cells
Trabecular bone was obtained from the iliac crest and from the proximal tibia used as donor sites in patients undergoing bone grafting procedures. The specimens were cut into small fragments 1-2 mm in diameter, thoroughly rinsed with Tyrode's solution, and cultured in 9 cm2 tissue culture plates containing ol-MEM with penicillin (33/~g/ml), streptomycin (50/~g/ml) and 10% fetal calf serum (6). After 2-3 weeks the culture plates were confluent with cells that had migrated from the trabecular bone. The cells were detached with trypsin (1 mg/ml) and seeded into 2 cm 2 multiwell culture dishes in which the subsequent experiments were performed. Photomicrographs of the cells were taken by placing the plastic multiwell dishes in the incubation chamber of an Olympus IMT-2 inverted microscope at 37~ The photomicrographs were taken using an Olympus 35 mm camera and A G F A PAN25 black and white film. Determination of Cyclic AMP Formation
Prior to the experiments, the cells were rinsed with Tyrode's solution and subsequently incubated for 30 min in serum free a~-MEM containing the cyclic AMP phosphodiesterase-inhibitor isobutylmethylxanthine (IBMX; 0.2mmol/1). The preincubation media were then removed and replaced with cr-MEM containing IBMX with PTH or vehicle. After 5 min, the incubation media were removed and cyclic AMP in the cell layer was extracted with 90% (v/v) n-propan-l-ol at 4~ for 24 hr. The extract was then evaporated, reconstituted with assay buffer and cyclic AMP was analyzed using a commerically available R I A kit with 125I-cAMP as tracer. Determination of PGE2-Formation Prior to experiments, the cells were rinsed with Tyrode's solution and preincubated in serum free a~-MEM for 30min. Thereafter the incubation
Bradykinin Induced Prostaglandin E 2 Formation in Human Osteoblasts
!23
medium was removed and fresh o:-MEM with test substances or vehicle was added for 5 rain. The incubation media were then withdrawn, acidified to pH 3.5 with HC1 and stored at - 2 0 ~ The amount of PGE2 synthesized was measured with RIA using a commercially available kit with ~25I-PGE2 as tracer. Statistic Evaluation Statistic evaluation of the data was performed using Student's t-test for unpaired samples. RESULTS Photomicrographs, taken of the human bone derived cells 24 hr after they had been seeded in 2 cm 2 multiwell culture dishes, demonstrated that the cells were cuboidal-shaped (Fig. 1).
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Fig. 1. Photomicrograph of isolated human osteoblast-like cells. Human osteoblast-hke cells obtained from trabeeular bone were seeded in plastic multiwell dishes 9 After 24 hr the dishes were placed in the incubator chamber of an Olympus IMT-2 inverted microscope and kept at 37 ~. Photomicrographs were taken using an Olympus 35mm camera. (• 190).
Ljunggren, Rosenquist, Ransj(i and Lerner
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Effectof bradykinin and des-arg9-bradykininon PGE2 formation and of PTH on cyclic AMP formation in isolated human osteoblast-like cells incubated for 5 min. Values represent means :t:SEM for 4 samples. **significantlydifferent from control p < 0.01, *significantlydifferent from control p < 0.05. Fig. 2.
Addition of the B2 bradykinin receptor agonist bradykinin (1/~mol/1; 5 min) to human isolated osteoblast-like cells caused a three-fold enhancement of PGE2 formation (Fig. 2). Similar results were obtained in three independent experiments using bone cells isolated from iliac crest as well as from proximal tibia. The B1 bradykinin receptor agonist des-argg-bradykinin (1/~mol/1; 5 min) also induced an increase in PGE2 formation. However, the effect of des-arg9-bradykinin on the PGE2 synthesis was significantly less than the effect of bradykinin at equimolar concentration (Fig. 2). Addition of PTH (10 nmol/1; 5 min) to isolated human osteoblast-like cells, preincubated in IBMX ( 0 . 2 m m o l / 1 ; 3 0 m i n ) caused a small, but significant increase in cyclic AMP formation (Fig. 2).
DISCUSSION
There is evidence that prostaglandins may be involved in inflammatory induced bone resorption in man in rheumatoid arthritis (7) and in periodontitis (8, 9). It is, however, unclear what causes the enhanced prostaglandin formation. The inflammatory mediator bradykinin is generated in areas of inflammation due to cleavage of kininogen by activated kallikrein (10). This nonapeptide is most
Bradykinin Induced Prostaglandin E2 Formation in Human Osteoblasts
125
well-known for its capacity to induce pain, cause vascular dilatation and enhance vascular permeability (11). We have recently shown that bradykinin is a potent stimulator of bone resorption in neonatal mouse calvaria by a mechanism that seems to be completely dependent on endogenous production of prostanoids (1, 2). Thus, bradykinin may be responsible for the enhanced levels of pros~ taglandins in inflammatory induced bone resorption. The data presented in this paper show that bradykinin causes a rapid formation of PGE2 in isolated human bone cells. The cells were cuboidal and responded to PTH (10 nmol/l) with an increase in cyclic AMP accumulation, similar to the observation made by MacDonald et al. (6). These findings and the observations that cells isolated from human bone, with the non-enzymatic technique used in the present study, are rich in alkaline phosphatase and synthetize osteocalcin in a manner regulated by PTH and 1,25(OH)2 vitamin D 3 (12, 13), indicate that the cells used express an osteoblastic phenotype. That an osteoblastic phenotype also includes the expression of bradykinin receptors, linked to prostaglandin formation, is suggested by the findings in the present report. This view is further supported by previous observations that bradykinin stimulates prostaglandin formation in isolated osteoblast-like cells from rat and murine calvaria, as well as in the murine osteoblastic cell line MC3T3-EI (4, 5). However, two cell lines derived from rat osteosarcomas (UMR 106-01 and ROS 17/2.8) do not respond to bradykinin with enhanced prostaglandin formation (4). In view of the findings in non-osteosarcoma osteoblasts, this may indicate that bradykinin induced prostaglandin formation is a feature mainly of nontransformed bone cells. Bradykinin (1 #mol/l) was a more potent stimulator of PGE2 formation in the human bone cells than des-arg9-bradykinin (1/~mol/1) was. This suggests the presence of B2 bradykinin receptors coupled to enhanced prostaglandin synthesis in human osteoblasts. Recently we have found that bradykinin, but not des-arg9-bradykinin, stimulates a rapid burst of prostaglandin-formation in mouse calvarial osteoblasts and in MC3T3-E1 cells (Ljunggren et al., in preparation). These observations and the fact that bradykinin induced prostaglandin formation in calvarial osteoblasts can be inhibited by a B2 bradykinin receptor antagonist (Ljunggren et al., in preparation), indicate the presence of B2 receptors for bradykinin in osteoblasts. The data in the present paper suggest that bradykinin induced prostaglandin formation in osteoblasts adjacent to inflammatory processes, and subsequent bone resorption, is a pathological phenomenon that occurs in man. ACKNOWLEDGEMENTS
This work was supported by grants from the Swedish Medical Research Council (B86-24X-07525-01A), the Swedish Association Against Rheumatic Diseases, the Royal 80 Year Fund of King Gustav V, the Swedish Dental Society, the Odontological Faculty, University of Ume~, Ume~, Sweden, Borgarskapets i Ume~ forskningsstiftelse, Stiftelsen Claes Groschinskys minnesfond and Magnus Bergvalls stiftelse.
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