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Interleukin-13 Inhibits Cell Proliferation and Stimulates Interleukin-6 Formation in Isolated Human Osteoblasts¹

Departments of Orthopedic Surgery (A.F.) and Internal Medicine (K.B.J., H.B., S.L., O.L.), University of Uppsala, S-751 85 Uppsala; and the Department of Internal Medicine, University of Goteborg (C.O.), S-41 345 Goteborg, Sweden

Address all correspondence and requests for reprints to: Anders Frost, M.D., Department of Orthopedic Surgery, University Hospital, S-751 85 Uppsala, Sweden. E-mail: anders.frost{at}ortopedi.uu.se

	Abstract

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Interleukin-13 (IL-13) is a recently identified cytokine that is secreted by activated T cells and regulates inflammatory responses. We have investigated the effects of IL-13 on isolated human osteoblast-like cells (hOB). IL-13 dose-dependently (1–100 pmol/L) reduced the incorporation rate of [³H]thymidine in hOB cells by more than 50%. Using a cell metabolic assay as well as direct cell counting, we found that treatment with IL-13 lead to a decrease in hOB cell number. The effect was both time and dose dependent, and after 12 days of culture, treatment with IL-13 (0.1 nmol/L) caused a 70% decrease in the number of cells. Also, IL-13 increased the levels of IL-6 messenger ribonucleic acid in hOBs, as measured by ribonuclease protection assay, and stimulated secretion of IL-6 into culture supernatants.

In conclusion, IL-13 inhibits cell proliferation and increases IL-6 formation in human osteoblasts. Our findings suggest that IL-13 may cause bone loss due to impaired osteoblastic growth and IL-6-induced osteoclast recruitment.

	Introduction

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REMODELING, the continuous turnover of mature bone, is a sequence of events including localized bone resorption followed by subsequent bone formation. This process is highly influenced by various inflammatory cytokines, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-6 (1). Disturbed regulation of the remodeling process, e.g. in metabolic bone diseases, could be a consequence of altered cytokine formation in the bone marrow (2). Also, systemic and local inflammatory diseases are often associated with pathology of bone, and inflammatory cytokines are therefore believed to be key regulators of bone cell activity.

IL-13, a recently cloned, T cell-derived cytokine, has been shown to be an important regulator of cells of the immune system (3). IL-13 is produced by T helper 2 (Th2) cells in response to antigen-specific activation and has pronounced effects on the cells of the monocyte/macrophage lineage (4). The Th2 subset of T cells produces several antiinflammatory cytokines (IL-4, IL-5, IL-10, and IL-13), and as unbalanced activation of proinflammatory cytokines, e.g. IL-1, TNF, and interferon-, could lead to the detrimental aspects of inflammation, the Th2 cytokines may act beneficially to control inflammation (5). IL-13 has, for example, been shown to inhibit the production of IL-1, IL-6, and TNF by activated monocytes and to enhance the synthesis of IL-1R antagonist (6, 7). The obvious coupling between inflammation and bone metabolism warrants a detailed study on the effects of IL-13 on bone cells. We therefore investigated the effects of IL-13 on proliferation and IL-6 production in primary isolated human osteoblasts-like cells (hOB).

	Materials and Methods

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Materials

MEM, trypan blue solution, and human recombinant IL-13, were purchased from Sigma Chemical Co. (St. Louis, MO). Penicillin and streptomycin (PEST), L-glutamine, trypsin-ethylenediamine tetraacetate (trypsin-EDTA), phosphate-buffered saline (PBS), and FCS were purchased from SVA (Uppsala, Sweden), and the Alamar Blue growth indicator was obtained from AccuMed (Westlake, OH). Human recombinant insulin-like growth factor I (IGF-I) was provided by Pharmacia-Upjohn (Stockholm, Sweden). [Methyl-³H]thymidine was purchased from Amersham (Aylesbury, UK). The IL-6 enzyme-linked immunosorbent assay (ELISA) was purchased from Pall Filtron (Solna, Sweden).

Isolation of hOB

Trabecular bone was obtained from the iliac crest of patients undergoing bone graft procedures. The specimens were cut to small fragments, 1–2 mm in diameter, thoroughly rinsed with PBS, and cultured in 75-cm² tissue culture flasks containing MEM supplemented with PEST (100 U/mL penicillin and 100 µg/mL streptomycin), amphotericin B (0.5 µg/mL), L-glutamine (2 mmol/L), and FCS (10%). After 3–4 weeks, the culture dishes were confluent with cells that had migrated from the trabecular bone. The cells were detached with trypsin-EDTA and seeded in multiwell culture dishes in which the subsequent experiments were performed as described below. Only first passage cells were used in these experiments. The project was approved by the local ethics committee.

Cell lines

The human osteosarcoma cell lines MG-63 and SaOS-II were obtained from American Type Culture Collection (Rockville, MD). These cells were cultured in MEM supplemented with 5% FCS, PEST, and 2 mmol/L L-glutamine.

Thymidine incorporation assay

hOB cells were seeded in 24-well culture plates at a density of 10,000 cells/well. They were left to adhere in MEM supplemented with 10% FCS and antibiotics for 24–48 h, after which the medium was changed to serum-free MEM. After 24 h of serum starvation, test substances were added in medium with 0.5% or 5% FCS, and 24 h later the cells were pulsed with 0.6 µCi [methyl-³H]thymidine for 24 h. Cells were harvested by trypsinization and transferred to a 96-well filter plate. The filters were washed, and the DNA was precipitated by ethanol before counting in a Wallac Microbeta (Wallac, Turku, Finland) liquid scintillation counter.

Alamar Blue proliferation assay

Osteoblastic cells were plated in 96-well culture plates at a density of 2000 cells/well in MEM containing 10% FCS and antibiotics. They were allowed to adhere for 24 h, after which a medium containing the experimental agents and 5% FCS was added, and the plates were incubated for different periods of time. Half of the medium was replenished every fourth day. At the end of the experiments, the medium was removed, and the cells were rinsed with PBS before DMEM, without phenol red or FCS, containing 10% Alamar Blue (vol/vol) was added. The wells were incubated with Alamar Blue solution for 5 h before measurements. The plates were measured with a fluorometer exciting fluorescence at a wavelength of 544 nm. The emitted light from each well was read at 590 nm. We have recently reported that the fluorescence thus obtained is directly proportional to the cell number (8). In some experiments cells were also detached with trypsin and counted in a hemocytometer after staining with trypan blue solution (0.4%).

Ribonucleic acid (RNA) isolation

Total RNA was isolated by the method of Chomczynski and Sacchi (9). Briefly, confluent hOB cells in 75-cm² culture flasks were washed in ice-cold PBS and lysed in a solution containing 4 mol/L guanidine thiocyanate, 0.5% sodium lauryl sarcosine, 25 mmol/L sodium citrate, and 0.7% ß-mercaptoethanol. The lysates were subjected to acid-phenol/chloroform extraction, and the RNA was precipitated with isopropanol and subsequently dissolved in H₂O. Before ribonuclease (RNase) protection assay analysis, the RNA was treated with 1 U deoxyribonuclease I for 60 min followed by proteinase K digestion and another phenol/chloroform extraction. The purified RNA was analyzed by agarose gel electrophoresis and quantified by spectrophotometry.

Probe

A 412-bp fragment of exons 2–5 of the human IL-6 gene was subcloned into a pCRII vector (10). A ³²P-labeled antisense RNA probe was transcribed with Sp6 RNA-polymerase from an XhoI-linearized plasmid. The human ß-actin probe was transcribed with Sp6 RNA polymerase from a linearized plasmid template obtained from Ambion (Austin, TX).

RNase protection assay

Total RNA (10 µg) was hybridized overnight with 140,000 cpm complementary RNA, IL-6, and ß-actin probe at 42 C in 20 µL buffer containing 80% formamide, 100 mmol/L sodium citrate (pH 6.4), 300 mmol/L sodium acetate (pH 6.4), and 1 mmol/L EDTA. Samples were digested with 200 µL RNase A and T1 solution for 45 min at 37 C. The RNases were inactivated by proteinase K treatment, and the samples were phenol/chloroform extracted, precipitated, and dissolved in gel loading buffer containing 80% formamide. The samples were electrophoresed on a 6% polyacrylamide-8 mol/L urea gel.

Measurement of IL-6 secretion

Osteoblastic cells were seeded in 24-well culture plates at a density of 10,000 cells/well. They were left to adhere in MEM supplemented with 10% FCS and antibiotics for 24 h, after which the medium was changed to serum-free MEM. Agonists were added, and the culture media were harvested after the experiments. IL-6 levels in the supernatants were analyzed by ELISA.

Test for endotoxin

The test media were tested for lipopolysaccharide contamination using the highly sensitive Limulus amebocyte lysate assay (11). The sensitivity of the assay is 0.03 endotoxin units/mL according to the manufacturer. No traces of lipopolysaccharide were detected in our incubation media (Hemachem Inc., St. Louis, MO).

	Results

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IL-13 inhibits hOB cell proliferation

We determined the effect of IL-13 on the rate of DNA synthesis by measuring incorporation of [³H]thymidine in hOB cells. A significant dose-dependent inhibition of [³H]thymidine incorporation was repeatedly demonstrated, with a threshold value at 1 pmol/L and a 50% decrease at 0.1 nmol/L. The inhibitory effect was similar regardless of whether the assay was performed in 0.5% FCS, as shown in Fig. 1, or in 5% FCS (data not shown). IGF-I (0.1 µmol/L), used as a positive control, stimulated [³H]thymidine incorporation to approximately 130% of the untreated control value in experiments conducted in 0.5% FCS (Fig. 1). To verify that the decrease in DNA synthesis also resulted in a reduction of hOB cell number in long term culture, we used the Alamar Blue proliferation assay, which we recently optimized for studies of proliferation in this cell system (8). IL-13 treatment dose and time dependently caused a decrease in cell metabolism, with a significant reduction detectable on day 8 and a decrease of about 25% after 12 days in culture. IGF-I (0.1 µmol/L) induced a significant stimulation of hOB cell number that reached 145% of the untreated control value after 12 days (Fig. 2). To clarify the changes in actual cell number over time, in both control wells and in wells incubated with 10 pmol/L IL-13, we counted the cells by use of a hemocytometer. In these experiments, IL-13 induced a decrease in the number of hObs after 12 days in culture compared to the initial number, whereas cell number steadily increased in control wells (Fig 3).

View larger version (27K): [in this window] [in a new window]   Figure 1. Dose-dependent inhibition of DNA synthesis in osteoblasts. Human osteoblast cells were seeded in 24-well culture plates at a density of 10,000 cells/well. Cells were left to adhere in MEM supplemented with 10% FCS and antibiotics for 24–48 h, after which the medium was changed to MEM with 0.5% FCS for 24 h before test substances were added in different concentrations. After 24 h, the cells were pulsed with 0.6 µCi [3H]thymidine for 24 h. Cells were harvested by trypsinization and transferred to a 96-well filter plate. The filters were washed, and the DNA was precipitated by ethanol treatment before counting in a Microbeta liquid scintillation counter. , IL-13; •, IGF-I. Values represent the mean ± SEM for six wells. The shaded bar represents the mean ± SEM for untreated controls. Similar data were obtained in three separate experiments.

View larger version (15K): [in this window] [in a new window]   Figure 2. Dose-dependent decrease in hOB cell number. Human osteoblast cells were plated at a concentration of 2000 cells/well in MEM containing 10% FCS and antibiotics. They were allowed to adhere for 24 h. Thereafter, medium containing the experimental agents and 5% FCS was added, and the plates were incubated for different periods of time. Half of the medium was replenished every fourth day. At the end of the experiments, the medium was removed, and the cells were rinsed with PBS before DMEM, without phenol red or FCS, containing 10% Alamar Blue (vol/vol) was added. The wells were incubated with Alamar Blue for 5 h, and fluorescence was measured as described in Materials and Methods. The fluorescence obtained in control wells was arbitrarily set at 100%, and the effects of agonists were compared to this value. , IL-13, day 4; , IL-13, day 8; , IL-13, day 12; •, IGF-I, day 4; , IGF-I, day 8; , IGF-I, day 12. Values represent the mean ± SEM for 12 wells. Similar results were obtained in three separate experiments.

View larger version (14K): [in this window] [in a new window]   Figure 3. Effect of IL-13 on hOB cell number. Human osteoblast cells were allowed to adhere for 24 h in 2-cm2 culture dishes. Thereafter, medium containing the experimental agents and 5% FCS was added, and the plates were incubated for different periods of time. Half of the medium was replenished every fourth day. Cells were detached at different times as shown, and cell number was calculated by counting the cells in a hemocytometer. Similar data were obtained in two separate experiments. , Control (Ctrl); •, IL-13.

As IL-13 is a known regulator of IL-6 synthesis in the inflammatory cascade, and IL-6 is a key regulator of bone cell activity, we determined the effects of IL-13 on IL-6 synthesis at both the RNA and protein levels in hOB cells. IL-13 time and dose dependently stimulated the accumulation of IL-6 protein in the cell supernatants. IL-13-induced IL-6 secretion was significantly different from that in untreated controls after 6 h (Fig. 4a), and the effect was seen at concentrations above 100 pmol/L (Fig. 4b). Also, using the RNase protection assay, we demonstrated that IL-13 up-regulated IL-6 messenger RNA (mRNA) levels in hOBs (Fig. 5). To extend the findings in hOBs regarding IL-6 formation, we investigated the effect of IL-13 on IL-6 secretion from two human osteosarcoma cell lines, MG-63 and SaOS-2. We could not detect any significant amount of secreted IL-6 when stimulating SaOS cells with IL-13. However, in MG-63 cells, IL-13 caused a small, but significant, increase in IL-6 secretion (Table 1).

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of IL-13 on IL-6 formation in human osteoblasts. hOB were seeded in 24-well culture plates at a density of 10,000 cells/well. Cells were left to adhere in MEM supplemented with 10% FCS and antibiotics for 24 h, after which the medium was changed to MEM with 0% FCS for 24 h before test substances were added in different concentrations. IL-6 was analyzed in the medium by ELISA. Values represent the mean ± SEM for six wells. A, Time course. •, IL-13; , control (Ctrl). B, Dose response. , IL-13; •, IL-1. The shaded bar represents mean ± SEM for untreated controls. Similar data were obtained in three separate experiments.

View larger version (60K): [in this window] [in a new window]   Figure 5. Effect of IL-13 on IL-6 mRNA levels in hOB cells. RNase protection assay in which 10 >µg total RNA isolated from hOB cells were hybridized with IL-6- and ß-actin-specific RNA probes. The samples were subjected to RNase treatment, and protected fragments were separated on a polyacrylamide gel. The autoradiograph above shows the IL-6 RNA hybrids at 412 bp and the corresponding ß-actin internal standard below. The autoradiographs were quantified on a phosphorimager, and the values, expressed as the ratio of the densities of IL-6 hybrids to the ß-actin hybrids, are given in parentheses below. Lane 1, GAPDH probe; lane 2, IL-6 probe; lanes 3 and 4, Untreated hOB cells (0.4% and 0.5%, respectively); lane 5, IL-13 (2 nmol/L; 8 h; 1.4%); lane 6, IL-13 (2 nmol/L; 16 h; 1.1%); lane 7, IL-1 (30 pmol/L; 8 h; 4.6%).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Bone loss in metabolic bone diseases is a consequence of an imbalance in the remodeling sequence, such that the coupled bone formation does not equal the preceding bone resorption. As proliferation of preosteoblasts is believed to be an important part of the bone-forming process, a cytokine that inhibits osteoblast proliferation would be expected to cause a negative remodeling balance, leading to subsequent bone loss. By using two methods representing different aspects of cell proliferation, thymidine incorporation to measure DNA synthesis and Alamar Blue reduction to indirectly measure cell number, we clearly demonstrate that IL-13 is a potent inhibitor of proliferation in phenotypically characterized isolated human osteoblasts. This was confirmed by direct cell counting. Our data showing that the absolute cell number decreases in IL-13-treated wells cannot be entirely explained by the demonstrated decrease in DNA synthesis. The clear reduction in cell number occurring after 8 days of treatment suggests either that IL-13 has effects on apoptotic cell death in hOBs, or alternatively, that in these cell cultures there is always a certain amount of programmed cell death and that the inhibition of cell proliferation therefore leads to a subsequent depletion in cell number. Furthermore, we recently demonstrated, in the same cell system, the proliferative actions of IL-1, TNF, and TNFß (15). The reported findings that IL-13 inhibits the secretion of IL-1 and TNF therefore indicate that the overall effect of IL-13 on human osteoblastic growth might be highly antiproliferative.

It is known that cells from the osteoblastic lineage regulate the formation and activity of osteoclasts, and evidence implicates osteoblast-derived cytokines in this process (16, 17). IL-6 is secreted from osteoblasts and stimulates the recruitment of osteoclast precursors from hematopoietic stem cells as well as the differentiation of these precursors into mature osteoclasts (18, 19, 20). IL-6 has also been postulated to be a paracrine mediator of estrogen actions on bone tissue. Hence, IL-6 production in the bone microenvironment is increased after estrogen withdrawal (2, 21). Considering the central role of IL-6 in bone turnover, we investigated the effect of IL-13 on IL-6 formation in osteoblasts. In contrast to the effect of IL-13 on cells from the monocyte lineage, where IL-13 inhibits IL-6 formation (6, 7), we found that IL-13 potently up-regulates IL-6 mRNA levels in hOBs and stimulates IL-6 secretion. Previous reports demonstrating up-regulation of IL-6 synthesis in cells not directly involved in inflammation, i.e. keratinocytes, glial cells, and endothelial cells, suggest cell-specific effects of IL-13 on IL-6 formation (22, 23, 24). Our findings in the human osteosarcoma cell line MG-63, in which IL-13 caused a small, but statistically significant, increase in the release of IL-6, further strengthen this view.

In conclusion, we have demonstrated that IL-13 inhibits cell proliferation and increases IL-6 formation in human osteoblasts. As IL-6 is known to be a potent stimulator of osteoclast recruitment, these findings clearly implicate IL-13 as a cytokine with the capacity to induce bone loss. The putative roll of IL-13 in the bone-remodeling sequence and whether IL-13 is involved in the pathogenesis of metabolic bone diseases are not known.

	Acknowledgments

 
We are grateful to Anna-Lena Johansson and Carolin Jönsson for skillful technical support.

	Footnotes

 
¹ This work was supported by grants from the Swedish Cancer Society, the Swedish Rheumatism Association, and the Swedish Society of Medicine.

Received November 17, 1997.

Revised April 21, 1998.

Revised June 3, 1998.

Accepted June 10, 1998.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Frost, A. Articles by Ljunggren, O. Search for Related Content PubMed PubMed Citation Articles by Frost, A. Articles by Ljunggren, O.