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In Vitro Secretion of Cytokines by Human Bone Marrow: Effects of Age and Estrogen Status¹

Department of Orthopedic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Julie Glowacki, Ph.D., Orthopedic Research, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115.

	Abstract

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It has been proposed that cytokines mediate the acceleration of bone loss following menopause. Because of the intimate relationship between bone marrow stromal cells and bone tissue, it is possible that marrow cells and their products contribute to the bone microenvironment and influence the regulation of bone cell differentiation and activity. We examined the production of cytokines by bone marrow stromal cells from a total of 37 women and 15 men undergoing total hip replacement for noninflammatory joint disease. Low-density mononuclear cells were isolated from bone marrow and were cultured in phenol red-free MEM medium supplemented with 10% FBS and antibiotics. Constitutive secretion of interleukin-6 (IL-6) was positively correlated with age in a series of 8 women and 5 men measured by bioassay (r = 0.98; P < 0.01) and in a series of 18 women and 10 men measured by immunoassay (r = 0.56; P < 0.01). The pattern of cytokine production by bone marrow stromal cells was examined in detail in 23 postmenopausal women, aged 49–88 yr. Basal secretion of immunoreactive IL-6 and IL-11, but not granulocyte-macrophage colony-stimulating factor, increased with time in culture. Exogenous IL-1ß stimulated secretion of IL-6 and IL-11 in a saturable, dose-dependent manner. Secretion of soluble IL-6 receptor was not correlated with secretion of IL-6, either constitutively or in the presence of IL-1ß. In 4 of 14 samples, IL-1ß also stimulated secretion of granulocyte-macrophage colony-stimulating factor. IL-1ß was undetectable in 7 of 9 cultures during the 2-week culture period. IL-6 did not stimulate secretion of IL-1ß in the 7 cultures tested. Cells were dependent upon serum for viability and growth and were not sustained by a serum substitute (1% insulin-transferrin-selenium-BSA). Cells grown in medium with 10% FBS and supplemented with 1% insulin-transferrin-selenium-BSA secreted 10-fold more IL-6 than cells grown in serum alone. Marrow from 7 women receiving estrogen replacement therapy showed lower constitutive secretion of IL-6 (75%; P < 0.006) and IL-11 (43%; P < 0.05) than marrow from age-matched controls and had blunted stimulation of IL-6 and IL-11 secretion by exogenous IL-1ß. These data indicate distinct patterns of cytokine production by human marrow stromal cultures dependent upon age and estrogen status.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
IT HAS BEEN proposed that tissue cytokines mediate the accelerated osteoclastic bone resorption that follows estrogen depletion (1, 2, 3). Although most information has been obtained from in vitro studies with murine bone marrow stromal cells, there are some data on cytokine production by human osteoblasts and bone marrow stromal cells. Cytokines, such as interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-11, are secreted by bone marrow stromal cells and affect the development and differentiation of immature hematopoietic cells (4, 5, 6). With estrogen deficiency or in inflammatory diseases, such as rheumatoid arthritis, increased secretion of these cytokines may promote the development of progenitors into osteoclasts and contribute to the associated pathophysiology of osteopenia (7, 8).

Human marrow stromal cells secrete a variety of cytokines that affect the development of hematopoietic stem cells. For example, it has been shown that IL-1ß stimulated the secretion of granulocyte- and macrophage-colony-stimulating factors (G-CSF and M-CSF) in human marrow stromal cells in long term culture (9). Similarly, IL-1 and tumor necrosis factor- (TNF-) stimulated G-CSF and GM-CSF secretion by human bone marrow stromal cell cultures and by clonal stromal-derived cells (10). Another human stromal fibroblastoid cell line (ST-1) dramatically increased its production of GM-CSF, G-CSF, and IL-6 messenger ribonucleic acid in response to IL-1 treatment (11). IL-1 and TNF- stimulated IL-6 secretion in simian virus 40-transformed human marrow stromal cells (12). Guba et al. (13) showed that human bone marrow stromal fibroblasts, defined by adherence to plastic, secreted GM-CSF and IL-6 in the absence of inflammatory cytokines. Seventy-two hours after the addition of human serum, IL-6 secretion was stimulated in bone marrow stromal cells.

There are controversies, however, about the nature of the cytokines secreted by human cells and their regulation by estrogens. Kurihara et al. (14) reported that recombinant human IL-6 (at concentrations of 10–100 pg/mL) stimulated formation of osteoclast-like mononuclear cells in 3-week bone marrow stromal cultures by inducing the release of IL-1ß. Schaafsma et al. (15) described that addition of an anti-IL-6 monoclonal antibody to cultures of human bone marrow stromal cells did not influence IL-1-induced colony-stimulating activity. Based upon these findings, they concluded that IL-6 does not induce CSF production by human marrow stromal cells or fibroblasts, and that IL-6 is not a cofactor in IL-1-induced CSF production by these cells. Additionally, Rifas et al. (16) reported that neither 17ß-estradiol nor progesterone modified IL-6 secretion that was induced by IL-1ß or TNF- in several strains of normal human osteoblasts and human bone marrow stromal cells. Others found that administration of estrogens modulated cytokine production by human bone cells in vitro. IL-1ß and TNF- stimulated the secretion of IL-6 in nontransformed human bone cells and that stimulation was partially reversed by the addition of 17ß-estradiol to the cultures (17). Also, estrogen was shown to inhibit IL-1ß and TNF--stimulated IL-6 secretion by a human fetal osteoblast cell line (hFOB/ER9) that expresses a high level of estrogen receptors (18).

To address these issues, we examined cytokine production in vitro by bone marrow stromal cells from postmenopausal women. We report a striking age dependence of IL-6 secretion by marrow cultures. Also, we report an increase in constitutive secretion of IL-6 and IL-11 that was dependent upon time in culture and was stimulated by treatment of the cultures with IL-1ß. Samples from subjects receiving estrogen replacement therapy (ERT) showed significantly lower cytokine secretion than those from age-matched women. In addition, we report difficulties growing cells under serum-free conditions and elevation in cytokine secretion induced by insulin-transferrin-selenium-BSA (ITS⁺)-supplemented medium.

	Materials and Methods

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Femoral bone marrow was obtained as discarded material from subjects undergoing total hip replacement for osteoarthritis. Subjects were excluded if they were taking medications or had comorbid conditions that could affect skeletal metabolism, including renal insufficiency, alcoholism, active liver disease, malabsorption, hyperthyroidism, rheumatoid arthritis, ankylosing spondylitis, or hyperparathyroidism. The marrow was suspended in Ca⁺²- and Mg⁺²-free phosphate-buffered saline (Life Technologies, Gaithersburg, MD) and passed through a 70-µm pore size cell strainer (Becton Dickinson VWR, Boston, MA). After collection by centrifugation for 10 min at 400 x g, cells were resuspended in phosphate-buffered saline. Low-density mononuclear cells were collected and washed after centrifugation on Ficoll-Histopaque 1077 (Sigma Chemical Co., St. Louis, MO). The percentage of colony-forming unit-stem cells was determined with a commercial human stem cell differentiation kit according to the manufacturer’s directions (Life Technologies). Depending upon the size of the surgical specimen, the yield of mononucleated cells ranged between 20–400 million/subject; thus, for some, limited numbers of studies were possible.

For culture, cells were resuspended in basal medium that consisted of phenol red-free MEM (Life Technologies) containing 10% heat-inactivated FBS (Life Technologies), 0.292 mg/mL L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Irvine Scientific, Santa Ana, CA). One million cells per 500-µL aliquot were seeded in triplicate in 24-well tissue culture plates (Falcon, Becton Dickinson VWR). After overnight incubation at 37 C in a humidified atmosphere of 5% CO₂ in air, 500 µL of either basal medium or treatment medium were added to each well. Recombinant IL-1ß and IL-6 were purchased from Genzyme (Cambridge, MA). ITS⁺ was purchased from Collaborative Biomedical Products, Becton Dickinson (Bedford, MA). Two days after the addition of medium, 700 µL conditioned medium were removed from each well and centrifuged at 400 x g for 3 min. The supernatants were stored at -80 C. Thereafter, 70% medium changes were made twice weekly.

In the majority of experiments, cytokine levels in conditioned medium were measured using commercially available enzyme-linked immunosorbent assay. Kits for IL-6, IL-1ß, and GM-CSF were obtained from Endogen (Cambridge, MA). Levels of detection were, respectively, 10.2, 6.7, and 10.2 pg/mL. IL-11 and soluble IL-6 receptor (sIL-6R) enzyme-linked immunosorbent assay kits were obtained from R&D Systems (Minneapolis, MN). Levels of detection were 15.6 and 31.3 pg/mL, respectively. Values were expressed as the mean ± SD for triplicate wells. Replicate cultures were terminated on day 2 for determination of the amount of DNA in each well, when available, by the method described by West (19).

In another series of experiments, 10⁶ cells were seeded directly in treatment media. Conditioned media were collected and assayed for IL-6 with a bioassay based upon the dependence of B9 cells on IL-6 for proliferation.

Values were compared using Student’s t test, when appropriate, or by the Mann-Whitney test for nonparametric data. Differences were considered significant when P < 0.05.

	Results

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Stem cells

The percentage of colony-forming unit-stem cells in marrow from nine men and women between 23–82 yr of age was 0.67 ± 0.17%, with no apparent differences with age and gender.

IL-6 bioactivity released by human marrow

In a series of 13 consecutive marrow cultures, IL-6 was measured by bioassay in media from marrow cultures after 2 days in vitro. There was a wide range in the amount of bioactive IL-6 secreted by human marrow. Constitutive secretion of IL-6 was correlated with the age of the subject for both women and men (Pearson correlation for the samples from women was r = 0.98; P < 0.01; Fig. 1A). None of these women was receiving ERT.

View larger version (16K): [in this window] [in a new window]   Figure 1. A, Constitutive secretion of bioassayable IL-6 (units per mL) by a series of 13 human marrow cultured for 2 days. The line represents the regression of the values from the women. B, Constitutive secretion of immunoassayable IL-6 (picograms per mL) by a series of 28 human marrow samples cultured for 2 days. Values at 10 pg/mL are below the detection limit. The line represents the regression of the values from the women.

Marrow cells from 28 consecutive subjects were incubated in basal medium overnight before further treatment, rather than incubated immediately with treatment medium. This modification ensured uniform seeding of all dishes. Seven sets of cultures were terminated after 2 days for the determination of total DNA content in each well. The basal levels of immunoreactive IL-6 were correlated with age for both women and men (Pearson correlation, r = 0.53; P < 0.01; Fig. 1B), as was the smaller series based upon bioassayable IL-6. None of these women was receiving ERT. Many of the cultures, especially those from the less elderly subjects, had undetectable immunoassayable IL-6 (<10.2 pg) under basal conditions (Table 1). The mean basal level of IL-6 ± SD for the women less than 59 yr of age was 13.6 ± 7.8 pg/mL, that for the women between 60–69 yr of age was 79.6 ± 43.6, and that for the women between 70–82 yr of age was 544 ± 190.0. There was little difference whether IL-6 was expressed on the basis of DNA content or of volume of conditioned medium.

IL-11, measured by commercial immunoassay, was undetectable in most samples of 2-day conditioned medium in 10 of 17 samples (Table 2). IL-11 was detected in most cultures at 9 days (Fig. 2). A modest age dependence was found for men (r = 0.7235; P = 0.018) and for estrogen-deficient women (r = 0.5365; P = 0.048).

View larger version (13K): [in this window] [in a new window]   Figure 2. Secretion of IL-11 by human marrow after 9 days in vitro. Samples were obtained from 9 men (+), 14 women (), and 10 women receiving ERT at the time of surgery (•). The line represents the regression of the values for estrogen-deficient women.

Bone marrow stromal cell cultures from 14 women and 9 men were continued for 12 days for demonstration of the kinetics of cytokine production under these conditions. Figure 3 shows representative patterns of cytokine secretion by marrow from a 72-yr-old woman. In all samples, constitutive IL-6 levels increased with time. Other cytokines exhibited different patterns. IL-11 secretion was undetectable in most samples after 2 days in culture, but was detectable after 6–9 days in vitro in most cultures (Table 2). GM-CSF was undetectable in all cultures. IL-1ß was undetectable in 7 of 9 cultures tested.

View larger version (17K): [in this window] [in a new window]   Figure 3. The effect of time in vitro on secretion of cytokines by bone marrow from a 72-yr-old woman.

IL-1ß stimulated secretion of IL-6 by human bone marrow stromal cultures whether expressed on the basis of volume of medium or DNA content in each well (Table 1). Stimulation of IL-6 secretion by IL-1ß ranged between 6- and 199-fold. The magnitude of stimulation for women less than 59 yr of age was 25 ± 5-fold, and that for women between 60–69 yr of age was 635 ± 426-fold; for women older than 70 yr whose basal levels were an order of magnitude higher than those of the younger women, IL-1 stimulated IL-6 secretion even further, with a mean of 10 ± 3-fold. In cultures of marrow from a 77-yr-old woman (Fig. 4), IL-1ß stimulated secretion of IL-6 at each time point. Constitutive secretion of sIL-6R was not correlated with secretion of IL-6, and IL-1ß did not stimulate secretion of sIL-6R.

View larger version (15K): [in this window] [in a new window]   Figure 4. Effects of IL-1ß (2 pmol/L) on secretion of IL-6 and sIL-6R (IL-6SR) by marrow from a 77-yr-old woman.

In a detailed experiment with marrow from a 72-yr-old woman, IL-1ß produced dose-dependent and saturable stimulation of IL-6 and IL-11 (Fig. 5, A and B). In addition, although 2 pmol/L IL-1ß did not stimulate GM-CSF secretion in cultures from all subjects, it was stimulatory in 4 of 14 cases (data not shown). In an experiment with marrow stromal cells from a 70-yr-old woman, IL-1ß caused a dose-dependent stimulation of GM-CSF secretion (Fig. 5C).

View larger version (15K): [in this window] [in a new window]   Figure 5. A, Effect of dose of IL-1ß on IL-6 secreted by human marrow from a 72-yr-old woman. B, Effect of dose of IL-1ß on IL-11 secreted by marrow from a 72-yr-old woman. C, Effect of dose of IL-1ß on GM-CSF secreted by marrow from a 70-yr-old woman. Values are shown for media collected after 2 days culture and are expressed as the mean ± SD for three wells. DNA was measured in the same wells.

We examined whether expansion of marrow cells influenced their subsequent response to IL-1ß. Time-course studies (e.g. Fig. 4) suggested a change in the magnitude of stimulation of IL-6 secretion depending upon the duration of culture in the continuous presence of IL-1ß. A set of marrow cells from a 79-yr-old woman was grown for 7 days in basal medium before treatment with IL-1ß. With time in culture, IL-6 and IL-11 levels increased in the cultures grown in either the absence or presence of IL-1ß (Table 3). In addition, the cells grown in basal medium for 7 days followed by 48 h of IL-1ß treatment showed a greater secretion of IL-6 and IL-11 than cells grown solely in medium with IL-1ß during those 9 days. Thus, these data show that cultures remain responsive to IL-1ß after expansion of the adherent marrow cells.

The feasibility of growing human marrow stromal cells in defined, serum-free conditions was assessed. Mononuclear cells from several marrow samples were cultured with serum-free basal medium supplemented with 1% ITS⁺, similar to serum-free conditions described by Long et al. (20). After initial attachment following overnight incubation, all cells detached from the plate during the next 48-h period in the absence of serum. The loss in viability was also reflected by low levels of IL-6 in conditioned medium (results not shown) compared to the constitutive IL-6 secretion by parallel cultures grown in medium supplemented with 10% FBS.

A series of seven marrow samples from women aged 51–75 yr was grown in medium with 10% FBS and 1% ITS⁺. Compared to the series cultured without ITS⁺ in which basal secretion was undetectable or low (Table 1; mean, 133 ± 189 pg/mL), the series with ITS⁺ secreted significantly more IL-6 (1733 ± 601 pg/mL; P < 0.001). Although all the cultures without ITS⁺ showed significant stimulation of IL-6 by IL-1ß (6- to 199-fold; Table 1), only one of the seven samples with ITS⁺ was further stimulated by IL-1ß (data not shown).

In a direct comparison of the effects of ITS⁺, marrow cells from a 66-yr-old woman were divided into either basal medium or medium supplemented with 1% ITS⁺ (Table 4). Constitutive secretion of IL-6 in ITS⁺-supplemented medium was 12-fold greater than that in basal medium whether based upon volume or DNA content (P < 0.001). In the absence of ITS⁺, IL-1ß stimulated IL-6 secretion 13-fold, but in the presence of ITS⁺, the stimulation by IL-1ß was only 1.4-fold. ITS⁺ stimulated the secretion of IL-11 in these cells and supported further stimulation of these cells by IL-1ß. Based upon these findings, cells were routinely grown in MEM with 10% FBS and without ITS⁺.

Cytokine secretion by marrow was evaluated with cells from nine women receiving ERT at the time of marrow procurement; each was compared to marrow from an age-matched woman and controlled for supplementation with ITS⁺ (Table 5). After 2 days in culture, marrow from women receiving ERT secreted approximately 7.5% of the amount of IL-6 secreted from the marrow of estrogen-deficient women (P < 0.006; Fig. 6A). Marrow cells from estrogen-deficient women secreted IL-6, which was stimulated between 2- and 44-fold by treatment with IL-1ß (P < 0.005). There was stimulation of IL-6 by IL-1ß in the samples from estrogen-replete women, but to only a fraction (10–50%) of that seen in the estrogen-deficient samples (Fig. 6B). After 2 days in cell culture, IL-11 secretion was significantly stimulated by IL-1ß in four cultures from estrogen-deficient women (Table 5). Marrow from estrogen-replete women did not secrete detectable amounts of IL-11 even in the presence of IL-1ß. By the ninth day in vitro, IL-11 was secreted by most cultures of marrow from estrogen-deficient women (Table 2 and Fig. 2). IL-11 secretion by marrow from estrogen-replete women (69 ± 109 pg/mL; n = 11) was 43% of the level secreted by marrow from estrogen-deficient women (163 ± 186 pg/mL; n = 16; P < 0.05; Fig. 2).

View larger version (25K): [in this window] [in a new window]   Figure 6. Effect of ERT on secretion of IL-6 by cultured human bone marrow. Samples were matched by age with samples from estrogen-deficient women. IL-6 was measured by immunoassay after 2 days culture under basal conditions as a function of age (A) and with and without 2 pmol/L IL-1ß (B). Points in A represent the mean ± SD for triplicate wells. Points in B represent the mean for triplicate wells and are paired for each subject.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
These results show that two determinants of bone metabolism, age and estrogen status, are also correlated with cytokine secretion by marrow in vitro. Use of marrow discarded during total hip arthroplasty for noninflammatory joint disease affords an opportunity to study marrow from well characterized subjects covering a wide age range. We previously reported a striking age-dependent increase in the formation of osteoclast-like cells from human marrow (21). Those observations may be explained by the data presented herein showing greater constitutive secretion of IL-6 in cultures of marrow from older than younger subjects.

It is likely that enhanced bone resorption in the estrogen-deplete state is due to multiple factors. Understanding of the role of marrow cytokines in osteoclastogenesis and as targets of estrogen regulation has been aided by studies with murine bone marrow stromal cells. A variety of factors, including IL-1ß, IL-3, IL-6, IL-11, GM-CSF, and TNF-, promote osteoclastogenesis in cultures of murine marrow (1, 22). Jilka et al. (23) showed that after ovariectomy, there was an increase in the number of colony-forming units for granulocytes and macrophages, in the number of putative progenitors of osteoclasts, and in the number of osteoclasts per bone. That increase was prevented by administration to the ovariectomized mice of 17ß-estradiol or an IL-6-neutralizing antibody. The media from marrow cultures from the ovariectomized mice had a significantly higher quantity of IL-6 than that from the sham-operated animals. In another study, murine bone cultures exhibited increased bone resorption when supernatants from bone marrow stromal cultures from ovariectomized mice were added. Those culture supernatants were found to contain increased IL-1ß bioactivity, measured as leukocyte-activating factor activity, as well as increased IL-6 protein and bioactivity (24). An additional study showed that IL-1ß and TNF were potent stimulators of IL-6 in bone marrow stromal cells and osteoblasts, an effect that was reversed by 17ß-estradiol and, to a lesser degree, by testosterone and progesterone (17). In contrast, Kitazawa et al. (25) reported that after ovariectomy, murine bone marrow stromal showed increased the secretion of IL-1ß and TNF, but not IL-6, and increased osteoclast formation. That increase was reversed by treatment with 17ß-estradiol, IL-1ß receptor antagonist, and TNF-binding protein, but not by an antibody to IL-6.

As in mice, estrogens may protect human skeletal mass by inhibiting cytokine secretion, although the exact mechanisms of this regulation in mice remain unknown (26). In the present study, marrow from women receiving ERT at the time of procurement behaved differently from marrow obtained from age-matched estrogen-deficient women. IL-6 secretion was always lower in the women receiving ERT, and IL-1ß stimulation of IL-6 was blunted. Additionally, these data suggest that estrogen modulation may include IL-11, previously believed to be independent of the estrogen status of the donor, although only murine cells were studied heretofore (26).

Our results are consistent with observations in a year-long longitudinal study in women that serum levels of IL-6 and sIL-6R were increased by 20% and 35%, respectively, after oophorectomy, but not after hysterectomy (27). Furthermore, in a cross-sectional study examining the effects of age and menopause on circulating levels of IL-6 and its soluble receptor in 145 healthy women (25–104 yr old), the same group of investigators found an exponential 10-fold increase with age in serum IL-6 and significantly higher levels of IL-6 and sIL-6R in postmenopausal women within 10 yr of menopause compared to those in premenopausal subjects (28).

Cohen-Solal et al. described kinetic patterns of cytokine secretion by marrow from postmenopausal women (29). They attributed cytokine release in marrow cultures to hematopoietic rather than stromal elements because the cytokine levels detected in those marrow cultures were comparable to those in peripheral mononuclear cell cultures. Under their culture conditions with autologous serum, IL-6, TNF-, and IL-1ß levels decreased with time in culture, findings attributed to monocyte maturation. Recently, that group reported that basal secretion of IL-1ß and TNF-, but not PGE₂ or IL-6, were inversely correlated with indexes of femoral neck longitudinal elasticity in nonosteoporotic women (30). Under the conditions described herein, constitutive secretion of IL-6 and IL-11 increased during the 2-week observation period. Expansion of the adherent cell layer was associated with greater secretion of IL-6 and IL-11 and robust stimulation of both by IL-1ß. Secretion of IL-1ß was detected in only two of nine cultures tested and at a much lower concentration than that reported by Cohen-Solal (29).

Bismar et al. examined cytokine production by bone marrow stromal cells from premenopausal, perimenopausal, and postmenopausal women, some of whom were receiving estrogen replacement therapy (31). Those investigators also found that cytokine secretion depended upon estrogen status. They reported that marrow from early postmenopausal women (<5 yr since menopause) and from women who recently discontinued ERT (after >8 yr of use) secreted higher amounts of IL-6, GM-CSF, and PGE₂ than marrow from premenopausal or late postmenopausal (>8 yr since menopause) women. They did not describe an age dependence of cytokine secretion with their immunoassays.

Some differences in results may be explained by technical differences. The conditions used in these studies support the growth and development of stromal elements from human bone marrow. We used only 10% FBS, whereas some studies used 20% autologous plasma (29), which may affect results due to its inherent variability in cytokine content, and other studies used 10% FCS, 15% FBS (5), 10–20% human serum (13), or 20% horse serum (14). Some of those studies (29) did not appear to use conditions that supported the viability of human marrow stromal cells.

The experiments with serum-free and ITS⁺-supplemented media show the necessity of establishing conditions that can support cell viability and the expansion of marrow cells without introducing artifacts. There have been several previous reports of human marrow cells grown in serum-free conditions. In a study examining the generation of osteoblast-like cells, bone marrow stromal cells were grown for 7 days in serum-free McCoy’s 5A medium supplemented with 1% ITS⁺ with no reported loss in viability (20). Lansdorp et al. also used serum-free conditions supplemented with insulin, transferrin, selenium, and albumin (32) for growing CD34⁺ human bone marrow stromal cells. It was our experience, however, that marrow stromal cells detached from the plate and lost viability within 36 h of the establishment of serum-free conditions. The elevation in IL-6 secretion in the presence of ITS⁺ is unexplained, but it is possible that insulin may regulate cytokine production. In preliminary studies, we found that constitutive secretion of IL-6 was greater in charcoal-stripped serum than in undiluted FBS (33). Those results suggested that serum contains a factor(s) that suppresses constitutive secretion of IL-6. Defined culture conditions are needed for rigorous and precise description of regulatory mechanisms in human cells.

The patterns of cytokine production by the cultures may provide insight into the nature of their production and the relationship between the hematopoietic and stromal cell populations. IL-6 seems to be a constitutive product, whereas IL-11 may be the product of a developed monolayer. Through paracrine or juxtacrine interactions, these cytokines can affect the hematopoietic cells in the cultures. IL-6 produced by marrow stromal cells has a paracrine role in regulating human myeloma cell growth (34). IL-6 and GM-CSF, among various cytokines, can support differentiation of hematopoietic progenitor cells into cells of the granulocyte-macrophage lineage (5, 6). The interaction between stromal cells and marrow progenitors may be mediated through direct cell to cell contacts because the formation of osteoclast-like cells is enhanced in cocultures of hematopoietic and marrow stromal cells. Physical separation of marrow mononuclear cells from the stromal cells by a Millipore membrane or addition of neutralizing antibodies to IL-6 and IL-1ß did not support the development of osteoclasts (35). Preliminary studies with adherent and nonadherent fractions of human marrow showed that IL-secreting and IL-1ß-sensitive cells were contained in the adherent or stromal cell fraction (33). Unfortunately, attempts to expand and subcultivate marrow stromal cells showed complete loss of constitutive IL-6 and IL-11 secretion and little stimulation by IL-1ß (33). Nevertheless, we report herein striking relationships between in vitro secretion of cytokines by primary outgrowths of marrow stromal cells and relevant clinical features of the marrow donor, i.e. age and estrogen status.

These data indicate that this in vitro system provides a reproducible method to describe differences in cytokine production by human marrow cultures while providing information about the impact of clinical features on marrow behavior in vitro. In a complementary study (36), we found that human marrow stromal cells secreted insulin-like growth factor I (IGF-I), IGF-binding protein-3 (IGFBP-3), and IGFBP-3 protease and that IGFBP-3 increased with age (r = 0.97; P = 0.0058). Marrow from a woman receiving ERT secreted only 38% of the IGFBP-3 that was secreted by cultures from two age-matched, estrogen-deficient women. Further, addition of 17ß-estradiol to cultured marrow from an estrogen-deficient woman suppressed IGFBP-3 secretion to 60% of the constitutive level. Thus, age and estrogen status modulated marrow secretion of elements of the skeletal IGF system. Finally, in that study, IL-1ß inhibited secretion of IGFBP-3 to either undetectable levels or levels between 11–35% of the control value. That IL-1ß inhibited marrow secretion of IGFBP-3 and stimulated marrow secretion of IL-6, IL-11, and, to some extent, GM-CSF suggests that IL-1ß has multiple paracrine effects on mediators involved in anabolic and catabolic aspects of bone metabolism.

	Acknowledgments

 
The authors thank Helen Reichel, Shahryar Noordin, Jeffrey Cohen, and Anne Lesieur-Brooks for assistance with some aspects of these investigations.

	Footnotes

 
¹ This work was supported by NIH Grants AG-12271 and AG-13519.

Received December 9, 1997.

Revised February 12, 1998.

Accepted February 18, 1998.

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