Boneand Mineral, Elsevier
14 (1991) 161-167
161
BAM 00384
Cytokine production by peripheral blood cells in postmenopausal osteoporosis
Maria T. Zarrabeitia, Jose A. Riancho, Jose A. Amado, Jose Napal and Jesus Gonzalez-Macias Departamento
de Medicina
Interna,
Hospital
M. Valdecilla,
Universidad
de Canrabria,
Santander,
Spain
(Received 4 February 1991) (Accepted 25 April 1991)
Summary
of
It has been suggested that the release cytokines with bone-resorbing activity from cells of the immune system might have a role in the pathogenesis of osteoporosis. We measured the secretion of the bone-resorbing products tumor necrosis factor, interleukin l/I and PGE, by peripheral blood mononuclear cells from seven healthy postmenopausal women and 12 patients with postmenopausal osteoporosis. No differences were observed between both groups either in unstimulated cultures or in cultures activated with calcitriol, endotoxin or phorbol esters. These results give no support for a role of peripheral blood immune cells in postmenopausal bone loss.
Key words: Osteoporosis; Interleukin-1; Tumor necrosis factor; Prostaglandin; Monocyte
Introduction
It has been suggested that the release of cytokines by cells present in the marrow adjacent to bone surfaces could be important in the regulation of bone remodelling [ 1,2].
Indeed, several secretory products of monocytes and macrophages, including prostaglandins, interleukin-1 (IL-l), and tumor necrosis factor (TNF) have potent effects on bone cells (for review, see Refs. 3-5). These products increase bone resorption and it has been hypothesized that they might be involved in the pathogenesis of osteoporosis and in the local bone loss associated with arthritis and periodontal disease [3]. In fact, some investigators have reported an increased spontaneous release of IL-1 by peripheral blood monocytes from osteoporotic patients [6]. How-
Correspondence to: Jose A. Riancho, Division of Endocrinology, University of Texas Health Science Center, San Antonio, TX 78284, USA. 0169-6009/91/$03.50@ 1991 Elsevier Science Publishers B.V.
162 ever those results have not been reproduced by others [7]. In an attempt to clarify this subject and to extend the observations to other secretory products of macrophages, we have studied the production of IL-l, TNF and prostaglandin E, (PGE,) by peripheral blood mononuclear cells (PBMC) from normal and osteoporotic women under different culture conditions.
Materials and Methods
Subjects
The control group consisted of seven postmenopausal healthy women, aged 65+4 years, They did not take any drug, and had a normal 20-parameter biochemical profile, including serum calcium, phosphate and alkaline phosphatase. The patient group consisted of 12 women (aged 63+6 years) with postmenopausal osteoporosis. They had no other diseases. All had at least one atraumatic vertebral fracture and a reduced bone mineral density (BMD), as measured with a Lunar dual X-ray densitometer (mean BMD, 0.73f0.08 g/cm*). They had not suffered fractures or received drug therapy (but analgesics) during the 2 months previous to the study. They received no drugs at all during the week prior to the study. Cell isolationand culture
Venous blood was obtained after overnight fasting and the mononuclear population was isolated by Ficoll gradient centrifugation. The cells were resuspended in RPM1 1640 medium (Flow, UK) supplemented with 10% heat-inactivated fetal calf serum, glutamine and antibiotics (here referred to as standard medium). Cells were seeded (5~ ld cells/ml) (1 ml per well) into 24-well plates (Costar, Cambridge, MA), and incubated for one hour at 37 “C in an atmosphere containing 5% CO,. Afterwards, the plates were gently agitated and the fluid was aspirated. In order to maintain a significant proportion of lymphocytes in the cultures (so mimicking the situations whereby the monokines may be produced in vivo), no further washing of the plates was performed. The population remaining in the wells comprised 60-75% monocytes, as assessed by esterase staining with a commercial kit (Sigma, St Louis, MO). Then, fresh standard medium with or without several agents was added to the wells. After incubating the plates for 24 h in the above-mentioned conditions, the supernatants were harvested and stored at -70 “C until assayed. The aliquots utilized to measure PGE, concentrations were stored in the presence of 10 &ml indomethacin. Preservative-free calcitriol (1,25dihydroxyvitamin D,, gift from Hoffmann-La Roche, Basel, Switzerland) was added to some cultures in a lo-’ M concentration. Endotoxin (LPS) from E. coli (Gifco, Detroit, MI) was added at a final concentration of 20 &r!~l. Phorbol 12-myristate 13-acetate (PMA) (Sigma, St Louis, MO) was added at a final concentration of 10 @ml.
163 Cytokine assays
PGE, was measured by RIA with a kit from New England Nuclear (Boston, MA). The cross-reactivity of PGE, in the system is 3.7%, while that of other cyclo-oxygenase products is less than 0.5%. Inter-assay coefficient of variation (CV) was 12%. PGE, was not analysed in PMA-activated cultures because of some technical problems. TNF was measured with a commercial ELISA that does not detect lymphotoxin (T Cell Sciences, Cambridge, MA). CV was 10%. IL-1 was measured by an IRMA (Medgenix, Belgium) which detects IL-l/? but not IL-la. CV was 7%. The results were analysed with the Mann-Whitney U-test for group comparisons, and the Spearman correlation coefficient.
Results
Calcitriol induced a marked increase in PGE, production, both in controls and in patients (P c 0.01). LPS also caused a slight increase in PGE,. No differences in PGE, secretion between patients and controls were observed in any culture condition (Fig. 1). LPS caused a significant increase in IL-1 and THF release (P c 0.03) in both groups of subjects. Calcitriol did not show any effect. PMA also enhanced IL-1 (P c 0.02) and TN,C (P < 0.03) secretion. We did not find significant differences in IL-1 or TNF production between pat,zats and controls in any culture condition (Figs. 2 and 3). In the patient group, the values of PGE, correlated with IL-l values, both in standard medium (r = 0.64, P C 0.05) and in the presence of calcitrio1 (r = 0.65, P c 0.05). There was no correlation between the age of the patients and cytokine production. There was no clear relationship between BMD and patterns of secretion. The correlation between BMD and IL-1 production in LPS-stimulated cultures was the only statistically significant one (r = -0.61, P C 0.05), but its biological significance is doubtful due to the high type I error probability when testPOE2 (nglml) 300
1
300
300
200-
200
200
0
I
0
0 0
100
r% -+g
QP
C BASAL
0L
0
CD
C
OP LPS
loo-
-f0
+-
0
8 0
O+C CALClTRlOL
Fig. 1. Secretion of PGE, by PBMC from control women (C) and osteoporotic women (OP), cultured in standard medium (Basal) and in media with LPS (20&ml), PMA (10 @ml) or calcitriol(lO0 nM). The horizontal lines represent the means. The number of PBMC isolated limited the culture conditions tested in some individuals.
164 IL-1 p (ng/ml) 150
_
c
c
OP
C
OP
BASAL
OP PMA
LPS
CALCITRIOL
Fig. 2. Secretion of IL-1 by PMBC from control women (C) and osteoporotic women (OF”)in different culture conditions (as in Fig. 1).
TNF (nglml) 30
0 10
ti co 0
lo-
10
0
0
0
8 0
0
0
30
30
30-
o
_j_ 1 o-
1.0
10
ab 0
0
8
0
1.0
1.o
8
8
0
0
0
77
0
T
0
0 0 0
Oi O.l-
0.1
C
OP BASAL
Fig. 3. Secretion
0.1
0.1
C
OP LPS
OP
C PMA
ofTNF by PBMC from control women (C) and osteoporotic
0
co
C
OP
CALCmRIOL
women (OP) in different
culture conditions (as in Figs. 1 and 2).
ing many possible relationships. Moreover, when the patients were divided into two groups according to BMD (higher or lower than 0.75 g/cm2), no differences in cytokine secretion were observed.
Discussion Several secretory products of immune cells have been shown to have potent effects on bone metabolism. Although also prodxed by some iymphocyte subsets, mono-
165 cytes are thought to be the main source of IL-l, TNF and PGE, among nucleated blood cells. Both IL-1 and TNF increase bone resorption, in vitro and in viva [1,3-51. PGE, has variable effects on collagen production and also increases osteoclastic bone resorption in vitro. Hence, the possibility has been raised that immune cells might have a role in the initiation and/or modulation of the cellular events taking place during bone remodeling [1,2]. It has been claimed that peripheral blood monocytes from osteoporotic patients release increased amounts of IL-l, as measured with a bioassay [6]. On the other hand, the treatment of postmenopausal women with estrogens may decrease IL-1 production by immune cells [8]. The same group has also recently reported a menopause-related increase in TNF secretion in healthy women [9]. These experiments represented an attractive model to study the pathogenetic mechanisms of osteoporosis, because blood cells are easier to obtain than bone cells. However, in the present study we have not been able to demonstrate any difference in IL-l, TNF or PGE, secretion between osteoporotic and healthy women. Our results are in agreement with a preliminary report from Hustmyer [7]. Differences in patient selection and experimental procedures could be responsible for these conflicting results. We used a mixed population in order to preserve the lymphocyte-monocyte interactions. And we studied freshly-isolated cells, so that the monocytes were stimulated by their adherence to plastic, a situation which might mimic the attachment to bone surfaces and which explains the high basal levels of cytokines and PGE, [lo]. We measured IL-1 and the other bone-resorbing factors by specific immunoassays, instead of the bioassays used in some previous reports [6,8]. Bioassays may have the advantage of being influenced by the release of inhibitors relevant to the effects of cytokines in vivo, but their relative unspecificity is a significant drawback. As in other studies on cytokine secretion by blood cells [ll], we observed large interindividual variations, Due to that variability and the relatively low number of subjects studied, we could have missed a small difference between the patient and control groups in some experimental condition. However, our negative results m each condition tested suggest that such a difference would be rather small and of marginal pathogenetic significance, if any, We do not have histomyrphometric data about our patients, but the biochemical indices of bone turnover were normal. Some patients with accelerated bone turnover may show an increased release of IL-1 [12]. Therefore, we cannot completely rule out a role of immune cells in some subsets of osteoporotic patients, particularly those with a high bone turnover rate. Our results do not mean that PGE and the cytokines are not involved in bone remodelling. They just show no differences in the production of these substances by peripheral blood cells from normal subjects and osteoporotic patients. Similar studies are’ worth to be repeated with cells obtained from the bone marrow, which may show different patterns of secretion, as suggested in another metabolic bone disorder, namely Paget disease [13]. In addition, osteoblast-produced cytokines are likely to have important paracrine effects in bone remodelling [14]. Under the cultsrc conditions used in this work, we did not find any effect of calcitriol on the secretion of IL-1 and TNF. Similar negative results have been reported in studies with osteoblastic cells [i4]. These results do not give support to the contention that the cyio-
166 some effects of calcitriol on bone [2]. Nevertheless, such hypothesis should not be rejected on the basis of our data. In fact, preliminary studies from GH.* laboratory indicate that the influence of calcitriol on cytokine secretion is variable, depending on the culture conditions, and specifically on the presence of other activating agents [15]. In conclusion, from our results it seems unlikely that different patterns of cytokine secretion between normal and osteoporotic subjects could be identified when studying peripheral blood cells. Although we cannot exclude a role of circulating immune cells in some particular subgroup of patients, studies more directly focused on the bone microenvironment are needed to investigate the pathogenetic role of cytokines in osteoporosis. kines gray mediate
Acknowledgements We thank Dr Gregory R. Mundy for reviewing the manuscript. This study was supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social.
References 1 Boyce BF, Aufdemorte TB, Garret IR, Yates AJP, Mundy GR. Effects of interleukin-1 on bone turnover in normal mice. Endocrinology 1989;125:1142- 1150. 2 Peck WA, Woods W. The cells of bone. In: Riggs BL, Melton LJ, eds. Osteoporosis: etiology, diagnosis, and management. New York: Raven Press, 1988. 3 Raisz, LG. Local and systemic factors in the pathogenesis of osteoporosis. N Engl J Med 1988;318:818-828. 4 Canalis E, McCarthy T, Centrella M. Growth factors and the regulation of bone remodeling. J Clin Invest 188;81:277-281. 5 Mundy GR. Local factors in bone remodeling. Ret Progr Horm Res 1989;45:507-531. 6 Pacifici R, Rifas L, Teitelbaum S, Slatopolski E, McCracken R, Bergfeld M, Lee W, Avioli LV, Peck VA. Spontaneous release of interleukin 1 from blood monocytes reflects bone formation in idiopathic osteoporosis. Proc Nat1Acad Sci USA 1987;84:4616-4620, 7 Hustmyer FG, Benninger L, Girasole G, Sakagami Y, Yu XP, Walker EB, Peacock M, Manolagas SC. Cytokine production and cell-surface marker analysis in blood mononuclear cells in osteoporosis. Calcif Tissue Int 1990;46(suppl2):A41. 8 Pacifici R, Rifas L, McCracken R, Vered I, McMurtry C, Avioli LV, Peck WA. Ovarian steroid treatment blocks a postmenopausal increase in blood monocyte interleukin 1 release. Proc Nat1 Acad Sci USA 1989;86:2398-2402. 9 Pacifici R, Brown C, Rifas L, Avioli LV. TNF alpha and GM-CSF secretion from blood monocytes in normal and osteoporotic women: a preliminary study on the effect of menopause and estrogen/progesterone treatment. Calcif Tissue Int 1990;46(suppl2):A57. 10 Hofsli E, Lamvik J, Nissen-Meyer J. Evidence that tumour necrosis factor (TNF) is not constitutively present in vivo. Stand J Immunol1988;28:435-441. 11 Schinder R, Mancilla J, Enders S, Ghorbani R, Clark SC, Dinarello CA. Correlations and interactions in the production of interleukind (IL-6), IL-l, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. B!osd 890;75:40-47. 12 kifici R, Rothstein M, Rifas L, Lau KHW, Baylink DJ, Avioli L, Hruska K. Increased monocyte interleukin-1 activity and decreased vertebral bone density in patients with fasting idiopathic hyper-
167 calciuria. J Clin Endocrinol Metab 1990;71:138-145. 13 Pioli G, Girasole G, Pedrazzoni M, Sansoni P, Erroi A, Davoli L, Ciotti G, Mantovani A, Passeri M. Spontaneous release of interleukin-1 (IL-l) from medullary cells of pagetic subjects. Calcif Tissue Int 1989;45:275-279. 14 Gowen M, Chapman K, Littlewood A, Hughes D, Evans D, Russell G. Production of tumor necrosis factor by human osteoblasts is modulated by other cytokines, but not by osteotropic hormones. Endocrinology 1990;126:1250-1255. 15 Zarrabeitia MT, Riancho JA, Olmos JM, GonzalezMacias J. 1,25_Dihydroxyvitamin D (1,25-OHD) reduces the production of tumor necrosis factor (TNF) by human blood monocytes. Calcif Tissue Int 1990;46(suppl. 2):A44.
14 (1991) 161-167
161
BAM 00384
Cytokine production by peripheral blood cells in postmenopausal osteoporosis
Maria T. Zarrabeitia, Jose A. Riancho, Jose A. Amado, Jose Napal and Jesus Gonzalez-Macias Departamento
de Medicina
Interna,
Hospital
M. Valdecilla,
Universidad
de Canrabria,
Santander,
Spain
(Received 4 February 1991) (Accepted 25 April 1991)
Summary
of
It has been suggested that the release cytokines with bone-resorbing activity from cells of the immune system might have a role in the pathogenesis of osteoporosis. We measured the secretion of the bone-resorbing products tumor necrosis factor, interleukin l/I and PGE, by peripheral blood mononuclear cells from seven healthy postmenopausal women and 12 patients with postmenopausal osteoporosis. No differences were observed between both groups either in unstimulated cultures or in cultures activated with calcitriol, endotoxin or phorbol esters. These results give no support for a role of peripheral blood immune cells in postmenopausal bone loss.
Key words: Osteoporosis; Interleukin-1; Tumor necrosis factor; Prostaglandin; Monocyte
Introduction
It has been suggested that the release of cytokines by cells present in the marrow adjacent to bone surfaces could be important in the regulation of bone remodelling [ 1,2].
Indeed, several secretory products of monocytes and macrophages, including prostaglandins, interleukin-1 (IL-l), and tumor necrosis factor (TNF) have potent effects on bone cells (for review, see Refs. 3-5). These products increase bone resorption and it has been hypothesized that they might be involved in the pathogenesis of osteoporosis and in the local bone loss associated with arthritis and periodontal disease [3]. In fact, some investigators have reported an increased spontaneous release of IL-1 by peripheral blood monocytes from osteoporotic patients [6]. How-
Correspondence to: Jose A. Riancho, Division of Endocrinology, University of Texas Health Science Center, San Antonio, TX 78284, USA. 0169-6009/91/$03.50@ 1991 Elsevier Science Publishers B.V.
162 ever those results have not been reproduced by others [7]. In an attempt to clarify this subject and to extend the observations to other secretory products of macrophages, we have studied the production of IL-l, TNF and prostaglandin E, (PGE,) by peripheral blood mononuclear cells (PBMC) from normal and osteoporotic women under different culture conditions.
Materials and Methods
Subjects
The control group consisted of seven postmenopausal healthy women, aged 65+4 years, They did not take any drug, and had a normal 20-parameter biochemical profile, including serum calcium, phosphate and alkaline phosphatase. The patient group consisted of 12 women (aged 63+6 years) with postmenopausal osteoporosis. They had no other diseases. All had at least one atraumatic vertebral fracture and a reduced bone mineral density (BMD), as measured with a Lunar dual X-ray densitometer (mean BMD, 0.73f0.08 g/cm*). They had not suffered fractures or received drug therapy (but analgesics) during the 2 months previous to the study. They received no drugs at all during the week prior to the study. Cell isolationand culture
Venous blood was obtained after overnight fasting and the mononuclear population was isolated by Ficoll gradient centrifugation. The cells were resuspended in RPM1 1640 medium (Flow, UK) supplemented with 10% heat-inactivated fetal calf serum, glutamine and antibiotics (here referred to as standard medium). Cells were seeded (5~ ld cells/ml) (1 ml per well) into 24-well plates (Costar, Cambridge, MA), and incubated for one hour at 37 “C in an atmosphere containing 5% CO,. Afterwards, the plates were gently agitated and the fluid was aspirated. In order to maintain a significant proportion of lymphocytes in the cultures (so mimicking the situations whereby the monokines may be produced in vivo), no further washing of the plates was performed. The population remaining in the wells comprised 60-75% monocytes, as assessed by esterase staining with a commercial kit (Sigma, St Louis, MO). Then, fresh standard medium with or without several agents was added to the wells. After incubating the plates for 24 h in the above-mentioned conditions, the supernatants were harvested and stored at -70 “C until assayed. The aliquots utilized to measure PGE, concentrations were stored in the presence of 10 &ml indomethacin. Preservative-free calcitriol (1,25dihydroxyvitamin D,, gift from Hoffmann-La Roche, Basel, Switzerland) was added to some cultures in a lo-’ M concentration. Endotoxin (LPS) from E. coli (Gifco, Detroit, MI) was added at a final concentration of 20 &r!~l. Phorbol 12-myristate 13-acetate (PMA) (Sigma, St Louis, MO) was added at a final concentration of 10 @ml.
163 Cytokine assays
PGE, was measured by RIA with a kit from New England Nuclear (Boston, MA). The cross-reactivity of PGE, in the system is 3.7%, while that of other cyclo-oxygenase products is less than 0.5%. Inter-assay coefficient of variation (CV) was 12%. PGE, was not analysed in PMA-activated cultures because of some technical problems. TNF was measured with a commercial ELISA that does not detect lymphotoxin (T Cell Sciences, Cambridge, MA). CV was 10%. IL-1 was measured by an IRMA (Medgenix, Belgium) which detects IL-l/? but not IL-la. CV was 7%. The results were analysed with the Mann-Whitney U-test for group comparisons, and the Spearman correlation coefficient.
Results
Calcitriol induced a marked increase in PGE, production, both in controls and in patients (P c 0.01). LPS also caused a slight increase in PGE,. No differences in PGE, secretion between patients and controls were observed in any culture condition (Fig. 1). LPS caused a significant increase in IL-1 and THF release (P c 0.03) in both groups of subjects. Calcitriol did not show any effect. PMA also enhanced IL-1 (P c 0.02) and TN,C (P < 0.03) secretion. We did not find significant differences in IL-1 or TNF production between pat,zats and controls in any culture condition (Figs. 2 and 3). In the patient group, the values of PGE, correlated with IL-l values, both in standard medium (r = 0.64, P C 0.05) and in the presence of calcitrio1 (r = 0.65, P c 0.05). There was no correlation between the age of the patients and cytokine production. There was no clear relationship between BMD and patterns of secretion. The correlation between BMD and IL-1 production in LPS-stimulated cultures was the only statistically significant one (r = -0.61, P C 0.05), but its biological significance is doubtful due to the high type I error probability when testPOE2 (nglml) 300
1
300
300
200-
200
200
0
I
0
0 0
100
r% -+g
QP
C BASAL
0L
0
CD
C
OP LPS
loo-
-f0
+-
0
8 0
O+C CALClTRlOL
Fig. 1. Secretion of PGE, by PBMC from control women (C) and osteoporotic women (OP), cultured in standard medium (Basal) and in media with LPS (20&ml), PMA (10 @ml) or calcitriol(lO0 nM). The horizontal lines represent the means. The number of PBMC isolated limited the culture conditions tested in some individuals.
164 IL-1 p (ng/ml) 150
_
c
c
OP
C
OP
BASAL
OP PMA
LPS
CALCITRIOL
Fig. 2. Secretion of IL-1 by PMBC from control women (C) and osteoporotic women (OF”)in different culture conditions (as in Fig. 1).
TNF (nglml) 30
0 10
ti co 0
lo-
10
0
0
0
8 0
0
0
30
30
30-
o
_j_ 1 o-
1.0
10
ab 0
0
8
0
1.0
1.o
8
8
0
0
0
77
0
T
0
0 0 0
Oi O.l-
0.1
C
OP BASAL
Fig. 3. Secretion
0.1
0.1
C
OP LPS
OP
C PMA
ofTNF by PBMC from control women (C) and osteoporotic
0
co
C
OP
CALCmRIOL
women (OP) in different
culture conditions (as in Figs. 1 and 2).
ing many possible relationships. Moreover, when the patients were divided into two groups according to BMD (higher or lower than 0.75 g/cm2), no differences in cytokine secretion were observed.
Discussion Several secretory products of immune cells have been shown to have potent effects on bone metabolism. Although also prodxed by some iymphocyte subsets, mono-
165 cytes are thought to be the main source of IL-l, TNF and PGE, among nucleated blood cells. Both IL-1 and TNF increase bone resorption, in vitro and in viva [1,3-51. PGE, has variable effects on collagen production and also increases osteoclastic bone resorption in vitro. Hence, the possibility has been raised that immune cells might have a role in the initiation and/or modulation of the cellular events taking place during bone remodeling [1,2]. It has been claimed that peripheral blood monocytes from osteoporotic patients release increased amounts of IL-l, as measured with a bioassay [6]. On the other hand, the treatment of postmenopausal women with estrogens may decrease IL-1 production by immune cells [8]. The same group has also recently reported a menopause-related increase in TNF secretion in healthy women [9]. These experiments represented an attractive model to study the pathogenetic mechanisms of osteoporosis, because blood cells are easier to obtain than bone cells. However, in the present study we have not been able to demonstrate any difference in IL-l, TNF or PGE, secretion between osteoporotic and healthy women. Our results are in agreement with a preliminary report from Hustmyer [7]. Differences in patient selection and experimental procedures could be responsible for these conflicting results. We used a mixed population in order to preserve the lymphocyte-monocyte interactions. And we studied freshly-isolated cells, so that the monocytes were stimulated by their adherence to plastic, a situation which might mimic the attachment to bone surfaces and which explains the high basal levels of cytokines and PGE, [lo]. We measured IL-1 and the other bone-resorbing factors by specific immunoassays, instead of the bioassays used in some previous reports [6,8]. Bioassays may have the advantage of being influenced by the release of inhibitors relevant to the effects of cytokines in vivo, but their relative unspecificity is a significant drawback. As in other studies on cytokine secretion by blood cells [ll], we observed large interindividual variations, Due to that variability and the relatively low number of subjects studied, we could have missed a small difference between the patient and control groups in some experimental condition. However, our negative results m each condition tested suggest that such a difference would be rather small and of marginal pathogenetic significance, if any, We do not have histomyrphometric data about our patients, but the biochemical indices of bone turnover were normal. Some patients with accelerated bone turnover may show an increased release of IL-1 [12]. Therefore, we cannot completely rule out a role of immune cells in some subsets of osteoporotic patients, particularly those with a high bone turnover rate. Our results do not mean that PGE and the cytokines are not involved in bone remodelling. They just show no differences in the production of these substances by peripheral blood cells from normal subjects and osteoporotic patients. Similar studies are’ worth to be repeated with cells obtained from the bone marrow, which may show different patterns of secretion, as suggested in another metabolic bone disorder, namely Paget disease [13]. In addition, osteoblast-produced cytokines are likely to have important paracrine effects in bone remodelling [14]. Under the cultsrc conditions used in this work, we did not find any effect of calcitriol on the secretion of IL-1 and TNF. Similar negative results have been reported in studies with osteoblastic cells [i4]. These results do not give support to the contention that the cyio-
166 some effects of calcitriol on bone [2]. Nevertheless, such hypothesis should not be rejected on the basis of our data. In fact, preliminary studies from GH.* laboratory indicate that the influence of calcitriol on cytokine secretion is variable, depending on the culture conditions, and specifically on the presence of other activating agents [15]. In conclusion, from our results it seems unlikely that different patterns of cytokine secretion between normal and osteoporotic subjects could be identified when studying peripheral blood cells. Although we cannot exclude a role of circulating immune cells in some particular subgroup of patients, studies more directly focused on the bone microenvironment are needed to investigate the pathogenetic role of cytokines in osteoporosis. kines gray mediate
Acknowledgements We thank Dr Gregory R. Mundy for reviewing the manuscript. This study was supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social.
References 1 Boyce BF, Aufdemorte TB, Garret IR, Yates AJP, Mundy GR. Effects of interleukin-1 on bone turnover in normal mice. Endocrinology 1989;125:1142- 1150. 2 Peck WA, Woods W. The cells of bone. In: Riggs BL, Melton LJ, eds. Osteoporosis: etiology, diagnosis, and management. New York: Raven Press, 1988. 3 Raisz, LG. Local and systemic factors in the pathogenesis of osteoporosis. N Engl J Med 1988;318:818-828. 4 Canalis E, McCarthy T, Centrella M. Growth factors and the regulation of bone remodeling. J Clin Invest 188;81:277-281. 5 Mundy GR. Local factors in bone remodeling. Ret Progr Horm Res 1989;45:507-531. 6 Pacifici R, Rifas L, Teitelbaum S, Slatopolski E, McCracken R, Bergfeld M, Lee W, Avioli LV, Peck VA. Spontaneous release of interleukin 1 from blood monocytes reflects bone formation in idiopathic osteoporosis. Proc Nat1Acad Sci USA 1987;84:4616-4620, 7 Hustmyer FG, Benninger L, Girasole G, Sakagami Y, Yu XP, Walker EB, Peacock M, Manolagas SC. Cytokine production and cell-surface marker analysis in blood mononuclear cells in osteoporosis. Calcif Tissue Int 1990;46(suppl2):A41. 8 Pacifici R, Rifas L, McCracken R, Vered I, McMurtry C, Avioli LV, Peck WA. Ovarian steroid treatment blocks a postmenopausal increase in blood monocyte interleukin 1 release. Proc Nat1 Acad Sci USA 1989;86:2398-2402. 9 Pacifici R, Brown C, Rifas L, Avioli LV. TNF alpha and GM-CSF secretion from blood monocytes in normal and osteoporotic women: a preliminary study on the effect of menopause and estrogen/progesterone treatment. Calcif Tissue Int 1990;46(suppl2):A57. 10 Hofsli E, Lamvik J, Nissen-Meyer J. Evidence that tumour necrosis factor (TNF) is not constitutively present in vivo. Stand J Immunol1988;28:435-441. 11 Schinder R, Mancilla J, Enders S, Ghorbani R, Clark SC, Dinarello CA. Correlations and interactions in the production of interleukind (IL-6), IL-l, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. B!osd 890;75:40-47. 12 kifici R, Rothstein M, Rifas L, Lau KHW, Baylink DJ, Avioli L, Hruska K. Increased monocyte interleukin-1 activity and decreased vertebral bone density in patients with fasting idiopathic hyper-
167 calciuria. J Clin Endocrinol Metab 1990;71:138-145. 13 Pioli G, Girasole G, Pedrazzoni M, Sansoni P, Erroi A, Davoli L, Ciotti G, Mantovani A, Passeri M. Spontaneous release of interleukin-1 (IL-l) from medullary cells of pagetic subjects. Calcif Tissue Int 1989;45:275-279. 14 Gowen M, Chapman K, Littlewood A, Hughes D, Evans D, Russell G. Production of tumor necrosis factor by human osteoblasts is modulated by other cytokines, but not by osteotropic hormones. Endocrinology 1990;126:1250-1255. 15 Zarrabeitia MT, Riancho JA, Olmos JM, GonzalezMacias J. 1,25_Dihydroxyvitamin D (1,25-OHD) reduces the production of tumor necrosis factor (TNF) by human blood monocytes. Calcif Tissue Int 1990;46(suppl. 2):A44.
