JOURNAL OF CELLULAR PHYSIOLOGY 15271-78 (1992)

Leukemia Inhibitory Factor/Differentiation-Stimulating Factor (LIF/D-Factor): Regulation of Its Production and Possible Roles in Bone Metabolism YOSHIKO ISHIMI, ETSUKO ABE, CHENC HE JIN,CHISATO MIYAURA, ME1 H U A HONC, M l D O R l OSHIDA, HISASHI KUROSAWA, YURl YAMACUCHI, M l K l O TOMIDA, M O T 0 0 HOZUMI, AND TATSUO SUDA* Department of Biochemistry, School of Dcntistry, Showa University, Tokyo 142 (Y.I.,E.A., C.H.]., C.M., M.H.H., T.S.); Department of Orthopedics, Tokyo Teishin Hospital, Tokyo 102 (M.O., H.K.); Department of Chemotherapy, Saitama Cancer Center Research Institute, Saitama 362 (Y.Y.,M.T., M .H.1, lapan Leukemia inhibitory factoridilferentiat ion-sti mu1 ati ng factor (LIF/D-factor), expression of its mRNA, and possible roles in bone metabolism were studied in murine primary and clonal osteoblast-like cells. Local bone-resorbing factors such as IL-1, TNFcr, and LPS strongly induced expression of LIFiD-factor mRNA in both clonal MC3T3-El cells and primary osteoblast-like cells. Neither parathyroid hormone nor la,25-dihydroxyvitamin D, stimulated cxpression of LIF/D-factor mRNA. LIFiD-factor per se did not stimulate expression of its own mRNA. Appreciable amounts of 1 IFiD-factor were detected in synovial fluids from rheumatoid arthritis (KA) patients but not in those with osteoarthritis (OA). Simultaneous treatment with LIFiD-factor, IL-1, and IL-6 at the concentrations found in synovial fluids from RA patients greatly enhanced bone resorption, though these cytokines did not stimulate bone resorption when separately applied. This suggests that LIF/D-factor produced by osteoblasts is in concert with other boneresorbing cytokines such as IL-l and IL-6 involved in the bone resorption seen in the joints of RA patienls. LIFiD-factor specifically bound to MC3T3-El cells with an apparcnt dissociation constant of 161 p M and 1,100 binding sitesicell. LIFiD-factor dose-dependently suppressed incorporation of ['Hlthymidine into MC3T3-EI cells. In addition, it potentiated the alkaline phosphatase activity induced by retinoic acid, though LIF/D-factor alone had no effect on enzyme activity. These results suggest that LIF/D-factor i s involved in not only osteoclastic bone resorption but also osteoblast differentiation i n conjugation with other osteotropic factors. 8 1992 Wiley-Liss, Inc.

The presence of a protein factor capable of inducing differentiation of murine myeloid leukemia cells (M1) was first reported by Ichikawa (1970) in conditioned media of various normal and tumor cell cultures. The factor was termed differentiation-stimulating factor (D-factor).In 1989, Lowe et a]. succeeded in cloning the cDNA of this factor. The amino acid sequence of D-factor was identical t o that of leukemia inhibitory factor (LIF) which had been cloned by Gearing et al. (1987) from conditioned media of murine Krebs I1 ascites tumor cell cultures. Subsequently, it was reported that LIFID-factor was identical to differentiation inhibitory activity (DIA) (Williams et al., 1988; Smith et al., 1988), human interleukin for DA cells (HILDA) (Moreau et al., 1988),hepatocyte stimulating factor-I11 (HSF-111) (Baumann and Wong, 1989), and cholinergic neuronal differentiation factor (Yamamori et al., 1989; Yamamori, 1991). These findings suggest that LIFID0 1992 WILEY-LISS, INC.

factor has a wide variety of biological activities in many tissues (Hilton and Gough, 1991). In a previous report, we showed that a differentiation-inducing factor(s) (DIF) for M1 cells is produced by Con A-stimulated murine spleen cells and that this factor induces bone resorption (Abe et al., 1986). Soon thereafter, Reid et al. (1990) reported that human and murine recombinant LIF stimulated bone resorption in neonatal murine calvaria. In contrast, Metcalf and Gearing (1989)reported a striking stimulatory effect of this cytokine on bone formation in mice injected with murine hemopoietic FD cells which had been transfected with an LIF expression vector. These findings

Received October 31, 1991; accepted February 3,1992. *To whom reprint requestsicorrespondence should be addressed.

72

ISHIMI ET AL

suggest that LIF plays a critical role in the regulation of both bone formation and resorption. It is known that LIFiD-factor receptors are localized preferentially in osteoblasts in bone (Allan et al., 1990). Several lines of evidence have indicated that osteoblasts are involved in not only bone formation but also bone resorption. We have previously shown that DIF activities separate into two major peaks with apparent molecular weights of 50,000 and 20,000 (50 kDa DIF and 20 kDA DIF) in the conditioned medium of a murine osteoblast-like cell line, MC3T3-El (Shiina-Ishimi et al., 1986; Abe et al., 1988). Both of these purified DIFs induced bone resorption (Abe et al., 1988; Ishimi et al., 1990). Although i t appears that the 50 kDa and 20 kDa DIFs are identical to the LIFiD-factor and IL-6, respectively, there is a t present no conclusive evidence for this assumption. I t has been reported that the levels of several cytokines, including IL-1 (Fontana et al., 1982), IL-6 (Hirano et al., 1988), and TNF (Buchan et al., 19881,are elevated in the synovial fluids of patients whose rheumatoid arthritis (RA) is complicated by severe bone loss. Recombinant IL-6 stimulated in vitro bone resorption in murine calvarial cultures when applied alone or in combination with IL-1 (Ishimi e t al., 1990). This suggests that IL-6 is somehow involved in bone resorption in the aforementioned RA patients. The present study indicates t h a t osteoblast-like cells express LIFiD-factor mRNA, preferentially, in response to local bone-resorbing factors such as IL-1, TNFa, and LPS. LIF/D-factor was detected in the synovial fluids of RA patients. Simultaneous treatment with LIFiD-factor, IL-1, and IL-6 a t the concentrations found in synovial fluids from RA patients induced bone resorption. MATERIALS AND METHODS Animals and drugs Sixteen-day-old pregnant ddy mice were obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan). Murine and human LIFiD-factor cDNAs were kindly supplied by Dr. Nishikawa, Kumamoto University (Kumamoto, Japan) and Dr. Goeddel, Genentech, Inc. (CAI, respectively. Recombinant human IL-la and IL- 1p were kindly provided by Dainippon Pharmaceutical Co. (Osaka, Japan) and Ohtsuka Pharmaceutical Co. (Tokushima, Japan), respectively. In assessing the activity of lymphocyte-activating factor (LAF), titers of IL-la and IL-1p were 1.5 x lo7 and 0.8 x lo7 U/mg of protein, respectively. Recombinant human LIFIDfactor was prepared by expressing its cDNA in CHO cells followed by purification (Lowe et al., 1989). Fifty units of LIFiD-factor were defined as the activity required to make 50% of M1 cells phagocytic. Using this assay system (Abe et al., 1986), the specific activity of LIFiD-factor was determined to be 5 x lo7 Uimg of protein. Recombinant human IL-6 was kindly supplied by Dr. T. Kishimoto, Osaka University (Osaka, Japan). The specific activity was determined to be 4.8 x lo6 UImg of protein using a n IL-6-responsive human B lymphoblastoid cell line, SKW-CL4 (Hirano et al., 1985). Human recombinant TNFa was obtained from Genzyme Co. (Boston, MA). LPS (prepared by the method of Westphal from Salmonella abortus equi) was

purchased from Difco (Detroit, MI). la,25-Dihydroxyvitamin D, (la,25(OH),D3) was purchased from PhillipsDuphar (Amsterdam, The Netherlands). Synthetic human parathyroid hormone [PTH(1-34)] was kindly supplied by Dr. M. Hori, Toyo Jozo Co. (Shizuoka, J a pan). PGE, and indomethacin were purchased from Sigma (St. Louis, MO). Cells and cell cultures A conal osteoblastic cell line, MC3T3-E1, established from newborn C57iBLi6 murine calvaria, was generously provided by Dr. Kodama, Ohu University (Fukushima, Japan). Primary osteoblast-like cells were isolated from the calvaria of newborn mice a s previously described (Ishimi et al., 1990). In short, twenty to thirty calvaria were collected and subjected to routine sequential digestions, using 0.1% collagenase (Wako Pure Chemicals, Osaka, Japan) and 0.2% dispase (Godo Shusei, Tokyo). Osteoblast-like cells collected in fractions 3 and 4 were combined. MC3T3-El cells and the primary osteoblast-like cells were cultured in a-MEM (Flow Laboratories, VA) supplemented with 10% FCS (J R Scientific, Woodland, CA). A murine myeloid leukemic cell line, M1, was cultured in Eagle’s minimal essential medium (Nissui Seiyaku Co., Tokyo), supplemented with double the usual concentrations of amino acids and vitamins (MVA) and 10% heat-inactivated calf serum (CS, GIBCO, Grand Island, NY). All cultures were maintained a t 37°C in a humidified atmosphere of 5% C 0 2 in air. Measurement of LIFID-factor mRNA Steady state levels of the LIFiD-factor mRNA were examined by Northern blot analysis using a murine LIFiD-factor cDNA probe labeled with [32PldCTP(specific activity, 3,000 Ciimmol; Amersham, Buckinghamshire, UK) (Ishimi et al., 1990). Total RNA was extracted from the cells with guanidine thiocyanate and purified by ultracentrifugation with a cesium chloride gradient. For Northern blot analysis, 20 pg of RNA were loaded and separated on a 1.5% agarose gel containing 2.2 M formaldehyde. The RNA was transferred to a nylon filter (Hybond N, Amersham) and hybridized with 32P-labeled cDNA (1 x lo6 cpmiml) in 50% formamide, 5 x SSPE (1 x SSPE is 0.15 M NaC110.01 M NaH,PO4/0.001 M EDTA-Na), 5 x Denhardt’s solution (1 x Denhardt’s solution is 0.02% polyvinylpyrrolidone/O.O2% Fico11/0.02% bovine serum albumin), 200 pgiml of denatured salmon testis DNA, and 0.1% SDS. After prehybridization for 3 4 h a t 42”C, hybridization was carried out overnight at 42°C and washed filters were exposed t o a n X-ray film (XAR-5; Kodak, Rochester, NY) for 24 h. Measurement of bone-resorbing activity The bone-resorbing activity was examined a s previously described (Shiina et al., 1986). Sixteen-day-old pregnant ddy mice were injected subcutaneously with 25 pCi of 45CaC1, (specific activity 1.8 Ciimmol; New England Nuclear Products, Boston, MA) and sacrificed 24 h later. The fetal calvaria were excised and divided into paired halves along the mid-sagittal suture. Each half was cultured for 24 h a t 37°C under 5% CO, in air in 0.5 ml of BGJb medium (GIBCO, Grand Island, NY)

73

LIF/D-FACTOR AND BONE

containing 1mgiml of bovine serum albumin (Fraction V; Sigma). After preculturing for 24 h, each half calvarium was transferred to fresh medium with (treated) or without (control) assay samples and bone-resorbing activity was calculated using the following formulae:

45Ca release (%) =

in medium 45Cain medium 45Cain bone 45Ca

x

100

+

46Ca

T/C ratio =

release (%) from

the treated calvaria 45Ca

release (%) from

the control calvaria Measurement of LIF/D-factor in RA synovial fluids The concentrations of LIFiD-factor in synovial fluids from patients with rheumatoid arthritis (RAj and osteoarthritis (OA) were measured by a radioreceptor assay using M1 cells. The synovial fluids were centrifuged for 10 min a t 1,200g to remove cell debris and then filtered through a membrane with a pore size of 0.2 pm. After the M1 cells (5 x lo6)suspended in 0.8 ml of Hepes-buffered growth medium had been incubated with 200 t ~ of l synovial fluid or graded concentrations of LIFiD-factor for 1 h a t 4"C, 1 ng (3 x lo5 cpm) of [12511LIF/D-factorwas added. Incubation was carried out for 2 h at 4°C and each reaction mixture was layered over 1 ml of FCS. Cell-associated and free L12511LIF/Dfactors were separated by centrifugation of the cells through FCS. The radioactivity in the cell pellet was counted in a gamma-counter. A typical displacement curve was obtained by adding 0.05-10 ng of human LIF/D-factor and the 50% inhibition of displacement was seen at 1 ng. In this assay, the intra-assay coefficient of variations was 10.9% and the inter-assay coefficient was 11.1%. LIFiD-factor receptor measurement MC3T3-El cells (5 x lo6) or murine primary osteoblastic cells were centrifuged and resuspended in Hepes-buffered a-MEM containing 10% FCS. After the cells had been preincubated either with (non-specific binding) or without (total binding) hundredfold unlabeled LIF/D-factor for 1 h at 4"C, graded amounts of [1251]LIF/D-factorwere added. Incubation was carried out for 2 h at 4°C. Cell-associated [1251]LIFiD-factorwas separated by the same procedure as the radioreceptor assay described above, and the radioactivity was counted in a gamma-counter. Specific binding of LIF/Dfactor to the receptor was calculated by subtracting the non-specific binding count from the total binding count.

FCS. The cells were pulse labeled with [3Hlthymidine (0.2 pciiwell; specific activity 80 Ciimmol; Amershamj for a final 24 h, and radioactivity in the TCA insoluble materials was counted.

Measurement of alkaline phosphatase activity MC3T3-El cells (2 x lo5) which had been precultured for 24 h were treated with LIMD-factor and/or retinoic acid for 5 days. After incubation, the cells were washed with Cat'- and Mg+'-free PBS [PBS(-)I, suspended in 0.05% Triton X-100 in PBS(-) containing 1 mM MgCl,, and sonicated for 30 sec. The homogenate was centrifuged at l,OOOg, and the supernatant was used for assay of alkaline phosphatase activity using p-nitrophenyl phosphate (PNP) as a substrate according to the method of Lowry et al. (1954). One unit of enzyme activity was defined a s the activity hydrolyzing 1nmol of the substrate per min per mg of protein. Statistical analysis Data were expressed as means SEM. The statistical significance was determined by Student's t test.

*

RESULTS LIF/D-factormRNA expression in clonal and primary osteoblastic cells treated with bone-resorbingagents Figure 1shows the time course of changes in steady state levels of LIF/D-factor mRNA in MC3T3-El cells cultured with either IL-la (A) or TNFa (B). After being cultured for 3 days, the cells were treated for the indicated times with 5 nglml of IL-la or 100 ngiml of TNFa. The LIF/D-factor mRNA transcript was detected predominantly as a 4 kilobase band. MC3T3-El cells which had been preincubated for 3 days did not express LIFiD-factor mRNA constitutively. However, LIFIDfactor mRNA expression occurred as early a s 1 h after addition of IL-la or TNFa, attained maximal levels at 3 h , and declined thereafter, Treatment of MC3T3-El cells for 3 h with 5 ngiml of IL-la, 10 nglml of IL-lp, 100 ng/ml of TNFa, and 5 pgiml of LPS significantly increased the expression of LIFiD-factor mRNA (Fig. 2A). Neither la,25(OH)2D3nor PTH induced expression of LIFiD-factor mRNA in MC3T3-El cells. No expression of LIFiD-factor mRNA was detected in cells treated with LIFiD-factor per se. Stimulation of LIFiD-factor mRNA expression by IL-la, TNFa, and LPS was observed not only in clonal osteoblastic MC3T3-El cells but also in primary osteoblast-like cells prepared from the calvaria of newborn mice (Fig. 2B). Treatment with bone-resorbing agents produced no increase in the expression of p-tubulin mRNA in either MC3T3-El cells or primary osteoblast-like cells (data not shown).

LIFiD-factor and other agents with DIF activity; cooperative effects on bone resorption Murine clonal cultures to which human recombinant Measurement of DNA synthesis in LIFiD-factor has been added exhibited bone-resorbing MC3T3-E1 cells activity in a dose-dependent manner, but the concenMC3T3-El cells were plated at 3 x lo3 cells per well tration required to induce in vitro bone resorption was and cultured for 24 h in a growth medium supple- about 100 times higher than that required to induce mented with 10% FCS. The cells were then treated with differentiation of M1 cells (DIF activity) (Fig. 3). Since the concentrations of LIFiD-factor indicated in Fig. 6A simultaneous treatment of M1 cells with the TdFlDfor 72 h in a growth medium supplemented with 1.5% factor and IL-1 has been reported to cooperatively in-

ISHIMI ET AL.

74

A

B

28s-

28S-

18S-

18s-

-z L

Q L c

5 -cn (P

c

C

a

L 9)

c

9)

E

.-M0

c.

C 9)

n

0

1

6 1 2 2 4

3

Time (hr)

Time (hr)

Fig. 1. Time course of changes in the expression of LIFiD-factor mRNA in MC3T3-El cells treated with either IL-lu or TNFor. After being cultured for 3 days, cells were exposed for the indicated times to 5 ngiml ofIL-la (A) or 100 nglml ofTNFa (B).Total RNA was extracted and subjected to Northern blot analysis as described in Materials and Methods

A 28s

18s

18s

2

3

4

5

6

7

1

2

3

4

5

6

7

8

9

-

B 28s

1

-

Pig. 2. Northern blot analysis of LIF/D-factor mRNA expression induced by various systemic and local bone-resorbing agents in osteoblast-like cells. A: The clonal MC3T3-El cells were treated for 3 h with vehicle (lane l),5 ngiml of IL-lor (lane 21, 10 ngiml of IL-lP (lane 3), 100 ngiml of TNFor (lane 41, 5 pgiml of LPS (lane 51, M PGE, (lane6), 5 ngiml of la,25(OH),D3 (lane7 ) ,100 ngiml of PTH (lane8 ) , or 20 ngirnl of LIFiD-factor (lane9). B Primary osteoblast-like cells prepared from the calvaria of newborn mice were treated for 3 h with vehicle (lane l),5 ng/ml of I L - l a (lane 21,100 ngiml ofTNFa (lane 31, 5 pgiml of LPS (lane4), 5 ng/ml of la,25(OH),D3 (lane 51,100 ngiml of PTH (lane 61, or 20 ngiml of LIFiD-factor (lane 7). Total RNA was extracted and subjected to Northern blot analysis as described in Materials and Methods.

duce M1 cell differentiation (Yamamoto-Yamaguchi et al. 1989), the combined effect of the two cytokines was also examined in a bone resorption assay. Simultaneous application of suboptimal doses of LIF/D-factor (6 ng/ml) and IL-la (0.03 ngiml) produced a cooperative effect on bone resorption which was partially inhibited by lop6 M indomethacin (Table 1).The dose-response curve of LIFD-factorstimulated bone resorption shifted to the left when 0.03 ngiml of IL-la was simultaneously

Human rLIFID-factor (ngiml)

Fig. 3. Various biological activities of recombinant human LIF/Dfactor. After murine myeloid leukemia cells (Ml) had been treated with graded concentrations of the LIFiD-factor for 3 days?phagocytic activity was measured as DIF activity (0 ' ' .o). Fetal murine calvaria were treated for 5 days with graded concentrations of LIFiD-factor without ( t . or) with 0.03 ngiml of IL-lu (Hor )1.25 x 10 M A23187 (A- A).Bone-resorbing activity was determined as described in Materials and Methods. Data are the means f SEM of six experiments.

added (Fig. 3). Similarly, the calcium ionophore A23187, another inducer of M 1 cell differentiation and also a macrophage activator, played a cooperative role in the LIFiD-factor-induced bone resorption (Fig. 3).

Measurement of LIF/D-factor levels in R A synovial fluids The concentrations of LIFiD-factor in synovial fluids from RA-affected joints were measured with a radioreceptor assay using M1 cells. The specimens collected from RA patients for use in this study satisfied the criteria of the American Rheumatism Association. No OA patients showed erosive bone changes. Seven out of

LIF/D-FACT0 TABLE 1. Synergistic effect of LIF/D-factor and IL-la on bone resorption'

Cytokines added LIF/D-factor ILla LIF/D-factor ILla LIF/D-factor IL-la Indomethacin

+

+ +

Exp. 1 6 ng/ml 0.03 ng/ml 6 ng/ml 0.03 ng/ml 6 ng/ml 0.03 ng/ml 10-fiM

0.94 f 0.08 1.19 k 0.11

m

Exp. 2

2.66 k 0.32'

+ 0.12 2.56 + 0.54'

1.66 f 0.213

1.76

1.09

* 0.27?

P < 0.05.

TABLE 2. Measurement of LIF/D-factor in synovial fluids from patients with RA and OA'

RA TW KH EM TM TS KS Mean i SEM

SI TY YT Mean

Age

Sex

RA RA RA RA RA RA RA RA

54 73

F F F F

~~

HW EW

AA SH

Diagnosis

+ SEM

44

RA

62 36 64 52 57 58

OA OA OA OA OA

34 33 72 74 64

F

M F F F F M F F F

IL-I

IL-6

LIF

IL-I

IL-1 . IL-1 . IL-6.

1 6 -:

LIF

+

+

+ IL-6

+

LIF

LIF Fig. 4. Cooperative effect of LIFiD-factor, IL-la, and IL-6 on bone resorption. Fetal murine calvaria prelabeled with 45Ca were treated for 5 days with 1 ng/ml of LIFiD-factor, 0.03 ng/ml of IL-la, and 20 ngiml of IL-6, separately or i n combinations of two of these. Data a r e the means i SEM of six samples. Significantly different from the groups treated with either LIFiD-factor, IL-la or IL-6 alone, or the groups treated with any combinations of two of these, "P < 0.05.

LIF/D-factor (ndml)

NU2 0.5

1.27 3.16 2.13 0.3 ND 0.83 0.57 0.97 ND 0.16 ND ND ND 0.03 f 0.03

*

'After M1 cells had been incubated with an aliquot of synovial fluid or graded concentrationsof unlabeled I.IF/D-factor for 1 h, 1 ng of f'"I]LIF/D-factor was added to the culture. Incubations were carried out for 2 h at 4°C. ZND:below the detection limit (< 0.1 ng/ml). :'Significantly different from OA patients.

nine specimens from RA patients showed significantly elevated levels of LIF/D-factor and its mean value in the nine patients was 0.97 2 0.35 ngiml (Table 2 ) . In contrast, none of the OA patients except patient TY had detectable levels of LIFiD-factor (Table 2). It has been reported that the concentration of IL-6 is greatly elevated in synovial fluids collected from patients with active RA (Hirano et al., 1988). In contrast, concentrations of IL-1, TNF, and PDGF in the affected joints of RA patients were relatively low (Buchen et al., 1987). Fontana et al. (1982) reported that the IL-1 concentrations in synovial fluids from patients with RA and OA were 0.48 % 0.04 Uiml (0.03 & 0.002 ngiml) and 0.07 0.04 Uiml (0.004 2 0.002), respectively, in terms of LAF activity. Furthermore, Hirano et al. showed that the IL-la and IL-1p levels were less than 0.1 ngiml in synovial fluids of 22 out of 25 RA specimens (unpublished data) and that the IL-6 level in RA patients was 15.6 i 13 ngiml (Hirano et al., 1988). In order to examine whether in situ levels of LIFiD-factor, IL-1, and IL-6 in synovial fluids from RA patients cause bone resorption, the cooperative effects of these three cytokines on in vitro bone resorption were examined.

*

Ib

T

;

1.11 10.14

'Fetal murine calsaria prelabeled with "Ca wele treated for 6 days with suboptimal concentrations of LIF/D-factm and ILla,separately or in combination, with or without indomethacin. Data are the means f SEM nf six samples. 'Significantly different from the groups treated with either IdF/U-factoror L l a , P < 0.05. 3Significantlydifferent from the group treated with both LIF/D-factor and ILla,

Patient

h

.-0

c

T/C ratio

LIFiD-factor (1ngiml), IL-la (0.03 ngiml), and IL-6 (20 ngiml) did not stimulate bone resorption when applied separately. However, simultaneously applying these three cytokines a t the same in situ concentrations had a highly stimulatory effect on bone resorption (Fig. 4). Analysis of LIFiD-factor receptors in osteoblastic cells Metcalf and Gearing (1989) reported that LIF stimulates not only bone resorption, but also bone formation in vivo. We therefore examined the binding of [1Z51]LIF/ D-factor to MC3T3-El cells. [1251]LIF/D-factorspecifically bound to the cells, and Scatchard analysis demonstrated 1,100 receptors per cell and a n apparent dissociation constant (kd) of 161 pM (Fig. 5). The maximal specific binding of L12511LIF/D-factor was similarly recognized in primary murine osteoblastic cells (murine primary osteoblastic cells, 7,100 cpm/106 cells; MC3T3-El cells, 9,500 cpmi106 cells), indicating the presence of LIFiD-factor receptors in primary osteoblastic cells. Effect of recombinant LIF/D-factor on osteoblastic function In order to examine whether the LIFiD-factor acts on osteoblasts as a n autocrine modulator, the effects of recombinant LIF/D-factor on DNA synthesis and alkaline phosphatase activity were examined in MC3T3-El cells. LIFiD-factor suppressed DNA synthesis in MC3T3-El cells a t a concentration as low as 0.1 ngiml, and inhibited DNA synthesis to 42% of the control level at 10 ngiml (Fig. 6A). LIF/D-factor did not affect alkaline phosphatase activity a t any of the concentrations tested (0.1-100 ng/ml), but did potentiate the enhancement of alkaline phosphatase activity induced by retinoic acid in MC3T3-El cells (Fig. 6B). DISCUSSION Recently, attention has been focused on the role of osteoblasts in osteoclastic bone resorption. The present

ISHIMI ET AL

76

We previously reported that the D-factor purified from conditioned media of Con A-treated murine spleen cells and L929 cells is capable of stimulating bone resorption (Abe et al., 1986).Thus, the experimental observation that LIF/D-factor is produced by osteoblasts in response to local bone-resorbing agents may explain, a t least in part, the role of osteoblasts in bone resorption. In a previous paper (Abe et al., 19881, we showed that MC3T3-El cells produced two DIFs with molecular weights of 50 kDa and 20 kDa during a differentiated stage spanning days 12 to 15 of incubation. In addition, the expression of LIFiD-factor and IL-6 mRNA occurred in MC3T3-El cells cultured for 15 days without any bone-resorbing agents (data not included). Thus, i t is reasonable to speculate that the 50 kDa and . = o 20 kDa DIFs produced by MC3T3-El cells are LIF/D0 100 200 300 400 factor and IL-6, respectively. 125 It should be noted t h a t factors such a s IL-1, TNF, Total I - LIF /D-factor added (cpm x 10 .3) IL-6, and LIFiD-factor, all of which induce differentiation of M1 cells into macrophages, belong t o a group of osteoclast activating factors (OAFS). This is in accord with the widely accepted concept that osteoclasts are derived from cells of the monocyte-macrophage lineage. It has been reported that any two-substance combina0.3 tion from the LIFiD-factor, G-CSF, IL-1 group induced differentiation of M1 cells more effectively than any of these cytokines alone (Yamamoto-Yamaguchi e t al., 1989).Onozaki et al. (1988, 1989) reported that simulgU 0.2 taneous treatment of M1 cells with IL-1 and either TNF or IL-6 induced differentiation synergistically. It is U noteworthy that the same cooperative effect was ob3 served in the bone resorption assay. We previously rem ported the cooperative effect of suboptimal doses of 0.1 IL-la and IL-6 on bone resorption (Ishimi et a]., 1990). Similarly, simultaneous treatment of murine calvaria with LIMD-factor and either IL-la or A23187, all of which have DIF activity, produced cooperative effects 0 on bone resorption (Fig. 3). Our results suggest that 10 20 30 40 50 LIFiD-factor acts a t different stages of osteoclast differBound lZ5l - LIF / D-factor ( pM ) entiation in the process of bone resorption, depending on whether the added compound is IL-la or A23187. Fig. 5. Binding of 11”5111JIF/I)-factorto MC3T3-El cells. A: Total Allan et al. (1990) reported that no specific binding of (a+), specific (A-A,, and non-specific (04) binding of [lzsIILIF/ D-factor to MC3T3-El cells at 4°C. B: Scatchard analysis of the spe- LIF/D-factor was detected in osteoclasts isolated from newborn rat long bone, indicating that the receptors for cific binding shown in A. LIF/D-factor are absent at least in mature osteoclasts. However, the possibility of the presence of LIFiD-factor receptors in osteoclast progenitors cannot be denied a t present. Osteoblasts have LIFiD-factor receptors a s study clearly demonstrates that the expression of LIFi well. Thus, i t is likely that LIFiD-factor induces osteoD-factor mRNA in osteoblasts occurs preferentially in clast differentiation either directly or indirectly by a response to local bone-resorbing agents such as IL-la, mechanism involving osteoblasts. IL-lp, TNFa, and LPS. To examine whether LIF/DIt is also likely that the cooperation of several cytofactor protein is produced along with its mRNA ex- kines occurs in the setting inflammatory bone resorppression in murine osteoblastic cells, we attempted to tion. IL-1, TNF, GM-CSF, PDGF, and IL-6 have been determine the mouse LIFiD-factor protein levels in con- detected in synovial fluids and conditioned media of ditioned media, but i t was unsuccessful. The cause of synovial cell cultures obtained from RA patients (Fonthis appeared to be due to the low sensitivity of murine tana e t al., 1982; Hirano et al., 1988; Buchan et al., LIFiD-factor. In the receptor assay using M1 cells, the 1988; Firestein and Zvaifler, 1987; Raines et al., 1989). binding affinity of murine recombinant LIFiD-factor Appreciable amounts of LIF/D-factor were also detected was only 1/50 as much a s that of human LIF/D-factor, in synovial fluids from patients with RA but not from though the former showed the same extent of bioactivi- those with OA (Table 2). Although it is not known ties as the latter in M1 cells. The reason for this discrep- which cells presented in the synovia of joints produce ancy between the binding activity and bioactivity is not LIFiD-factor, i t is likely that osteoblasts a s well as activated lymphocytes participate in the production of LIF/ clear.

A

[.