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Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8020
Address all correspondence and requests for reprints to: Karl Insogna, M.D., Section of Endocrinology, Department of Medicine, Yale University School of Medicine, 333 Cedar Street, FMP 106, P.O. Box 208020, New Haven, Connecticut 06520-8020. E-mail: karl.insogna{at}yale.edu
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Abstract |
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Introduction |
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Several in vitro experiments have established that both IL-6 and IL-11 also play prominent roles in bone homeostasis and share similar functions in that tissue as well. First, both molecules are produced by osteoblasts in response to osteotropic factors such as PTH and 1,25-dihydroxyvitamin D (6, 7). Transcripts for both the IL-6 and IL-11 receptors are expressed in osteoblast progenitor cells and mature osteoblasts (8). In vitro, IL-6 and IL-11 stimulate osteoclast formation, which appears to be mediated by the signal transducer gp130 (9). In addition, recent data suggest that IL-11 inhibits osteoclast apoptosis (10, 11). In the aggregate, these data suggest a proresorptive action for both cytokines in bone.
Recent studies have also indicated a role for these cytokines in states of disordered mineral homeostasis. Thus, neutralizing IL-6 in vivo prevents the increased osteoclastogenesis that occurs after estrogen withdrawal (12). In states of PTH excess, such as primary hyperparathyroidism, circulating levels of IL-6 are elevated and correlate to markers of bone resorption (13). In vivo, neutralizing IL-6 attenuates PTH-induced bone resorption in mice (14). Reduced expression of IL-11 in the bone marrow of the senescence-accelerated mouse (SAMP6) is associated with diminished bone formation and osteoclastogenesis; the former is thought to explain in part the reduced bone mass seen in these animals (15).
Although both IL-6 and IL-11 appear to have important roles in mediating bone resorption, studies addressing the coordinate regulation of these molecules by osteotropic hormones have not been reported. We therefore explored the responses of both cytokines to alterations in parathyroid function. Our data indicate that IL-6 suppresses PTH- induced IL-11 production. As both IL-6 and IL-11 stimulate osteoclast formation, down-regulation of IL-11 by IL-6 may help modulate the resorptive response to PTH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurement of circulating levels of IL-11 in
hyperparathyroidism
Sera were collected from 22 normal volunteers, 29 patients with
primary hyperparathyroidism, and 7 patients with hypoparathyroidism.
The patients with hypoparathyroidism have been previously described
(13).
Effect of parathyroidectomy on cytokine levels
Sera from three patients who underwent parathyroidectomy were
obtained before surgery and every 4 h thereafter for 24 h.
Mean serum calcium in these patients fell from a preoperative value of
10.7 to 9.7 mg/dl 24 h postoperatively. All patients have
subsequently remained eucalcemic. All samples were frozen immediately
at -70 C until analyzed.
Animal studies
Animal experiments were approved by the Yale animal care and use
committee and were conducted in accordance with the NIH Guide for the
Care and Use of Laboratory Animals.
Baseline levels of IL-6 and IL-11
Six-week-old CD-1 mice (Charles River Laboratories, Inc., Wilmington, MA) were used in all studies. Baseline
circulating levels of IL-6 and IL-11 were determined in samples
obtained from eight mice. Six-week-old female IL-6 knockout mice
(13) (IL-6-/-; n = 6) and
their wild-type littermates (IL-6+/+; n = 6)
were anesthetized, and blood was collected by cardiac puncture. Samples
were stored at -70 C until analyzed.
PTH infusion
Rats. Twelve-week-old Sprague Dawley rats (250 g;
Taconic Farms, Inc., Germantown, NY) were infused with
human PTH-(1–84) [hPTH-(1–84)] at 12.9 pM/h for 5
d as previously reported (14). During the infusion, serum
samples were collected daily by tail bleed. Animals infused with
vehicle were studied in parallel. Samples were stored at -70 C until
analyzed.
Mice. PTH infusions were performed as previously reported
(14). hPTH-(1–84) was infused at a rate of 4.3
pM/h for 5 d using osmotic minipumps (Alza Corp., Palo Alto, CA), then mice were anesthetized, and blood
was collected by cardiac puncture. Samples were stored at -70 C until
analyzed. Just before infusion, five mice were injected with IL-6-
neutralizing antibody (1 mg/mouse, ip) as previously described
(14). Five mice received an irrelevant isotype-matched
antibody (clone RR8-1 to the V
11.1 and V 11.2 murine T cell
receptors, which are not expressed in CD-1 mice). Five mice received
hPTH-(1–84) infusions alone.
Studies in cultured cells
Human osteoblast-like cells (SaOS-2) were cultured in RPMI
medium (Life Technologies, Inc., Gaithersburg, MD)
supplemented with 10% FCS (Sigma, St. Louis, MO) and 1%
penicillin/streptomycin (Sigma). The cells were grown to
confluence in six-well plates. At confluence, media were aspirated and
replaced with 2 ml medium containing 0.4 mg/well (final concentration,
0.2 mg/ml) neutralizing antibody to human IL-6 (R & D Systems, Inc., Minneapolis, MN) or nonimmune antiserum (goat antirabbit
antibody, R & D Systems, Inc.). hPTH-(1–84)
(Bachem, King of Prussia, PA) was then added to each well
at a final concentration of 10 nM. Conditioned media were
collected 24 h later, stored at -70 C and analyzed for
concentrations of IL-6 and IL-11. Duplicate measurements were performed
on conditioned media from each well, and the entire experiment was
repeated three times. Primary murine and human osteoblasts were
provided by Dr. Mark C. Horowitz (Cell Core, Yale Core Center for
Musculoskeletal Disorders, New Haven, CT) and isolated and cultured as
previously described (16, 17). Experiments examining the
effects of neutralizing antiserum to IL-6 on PTH-induced IL-11
production in primary human and murine cells were conducted exactly as
described above for the SaOS-2 cells. For the experiments in murine
cells, a neutralizing antibody to murine IL-6 (R & D Systems, Inc.) was used.
Cytokine assays
Concentrations of human IL-6 and IL-11 were measured by solid
phase ELISAs (R & D Systems, Inc.). The lower limits of
detection for these assays are less than 1 pg/ml for IL-6 and less than
8.0 pg/ml for IL-11. The precision of these measurements in our
laboratory is: for IL-6: intraassay coefficient of variation (CV),
4.4%; interassay CV, 5.4%; and for IL-11: intraassay CV, 3.9%;
interassay CV, 5.1%. Rat and mouse IL-6 were measured using a murine
solid phase ELISA (R & D Systems, Inc.). We routinely
increased the antibody incubation time to increase the sensitivity to
3.9 pg/ml. The precision of this measurement in our laboratory is:
intraassay CV, 3.2%; and interassay CV, 4.1%.
A murine sandwich ELISA was developed to measure murine IL-11 in cell culture supernatant and serum. Goat antimouse IL-11 (R & D Systems, Inc.) was diluted to a working concentration of 2 µg/ml in PBS without carrier protein. Ninety-six-well microplates (Costar, Corning, Inc., Corning, NY) were immediately coated with 100 µl/well of the diluted antibody. The plate was sealed and incubated overnight at room temperature or in an incubator at 25 C. Each well was then aspirated and washed with 350 µl wash buffer (0.05% Tween 20 in PBS, pH 7.4). The process was repeated for a total of four washes. The wells were blocked with 300 µl block buffer (1% BSA and 5% sucrose in PBS with 0.05% sodium azide) for a minimum of 1 h at room temperature. The aspiration/wash cycle was then repeated.
A seven-point standard curve using a 2-fold serial dilution of mouse IL-11 (R & D Systems, Inc.) was diluted in reagent diluent: 0.1% BSA and 0.05% Tween 20 in Tris-buffered saline (20 mM Trizma base and 150 mM NaCl), pH 7.3 (0.2 µm pore size filtered). Standards ranged from 7.8–500 pg/ml.
One hundred microliters of the reagent diluent were added to each well, followed by the addition of 100 µl of either standard or sample. After a 2-h incubation, the plate was washed as before. Two hundred microliters of biotinylated goat antimouse IL-11 (200 ng/ml) were then added, followed by a 2-h incubation at room temperature. The aspiration/wash cycle was repeated as described above. One hundred microliters of streptavidin-horseradish peroxidase (R & D Systems, Inc.) were then added to each well, followed by a 20-min incubation at room temperature. The aspiration/wash cycle was repeated as described above, followed by the addition of 100 µl of equal volumes of hydrogen peroxide and tetramethylbenzidine. After a 30-min incubation at room temperature, the reaction was stopped with 50 µl 2 N sulfuric acid. The OD of each well was determined using a microplate reader set at 450 nm, and at 540 nm for wavelength correction. Calculations are performed using a computer-generated 4PL curve fit. The sensitivity of the assay is 2.9 pg/ml. The intraassay coefficient of variation is 3.7%, and the interassay coefficient of variation is 4.7%.
PTH assays
Determination of serum hPTH-(1–84) by immunoradiometric assay
and measurement of circulating levels of midmolecule PTH were
determined as previously reported (13).
Northern analysis
For Northern blot analyses, SaOS-2 cells were grown to
confluence in 10-cm diameter tissue culture dishes and treated with 10
nM hPTH-(1–34) for 0, 2, 4, 6, and 8 h to establish
the time of maximal PTH-induced increase in IL-11 mRNA expression.
After establishing that 2 h was optimal, the experiment was
repeated using the following conditions. Cells were treated for 2
h with 1) neutralizing antibody to IL-6 (0.2 mg/ml, final
concentration), 2) nonimmune serum (0.2 mg/ml, final concentration), 3)
hPTH and neutralizing antibody to IL-6, or 4) hPTH and nonimmune serum.
The experiment was repeated four times. mRNA was isolated using the
TRIzol reagent, electrophoresed, and transferred to a nylon membrane
using the method of Church and Gilbert (18). The IL-11
probe was provided by Dr. Paul Schendel (Genetics Institute, Boston, MA) (19, 20). Expression of mRNA
for murine GAPDH (probe from Ambion, Inc., Austin, TX) was
used as a loading control.
Statistical analyses
All analyses were performed using the Systat statistical package
(version 5.2.1, Systat, Inc., Evanston, IL). Comparisons of data from
patients before and after parathyroid surgery and from the rat infusion
studies were performed using repeated measures ANOVA. All other
comparisons were performed using t test for unpaired data.
Data are presented as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mean fasting serum IL-11 values were suppressed in patients with
hyperparathyroidism compared with euparathyroid controls or subjects
with hypoparathyroidism. Thus, in 22 controls the mean circulating
IL-11 value was 12.4 ± 1.0 pg/ml, significantly higher than the
mean value of 5.7 ± 1.2 pg/ml in 29 patients with primary
hyperparathyroidism (P < 0.001). Subjects with
hypoparathyroidism had mean serum IL-11 values no different from
controls (10.9 ± 1.0 pg/ml). As expected, based on our prior
studies, IL-6 levels were elevated in the hyperparathyroid patients and
suppressed in the subjects with hypoparathyroidism compared with
eucalcemic controls (controls, 1.1 ± 0.1 pg/ml; hyperparathyroid
subjects, 12.0 ± 1.4; hypoparathyroid subjects, 0.4 ± 0.04;
P < 0.001 controls vs. hyper- and
hypoparathyroid subjects). Figure 1A
summarizes the pattern of cytokine expression in the three groups of
study subjects. In euparathyroid and hypoparathyroid individuals IL-11
circulates in concentrations higher than those of IL-6, whereas in
hyperparathyroid subjects the relationship is reversed.
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, there was a strong negative correlation between circulating
levels of PTH and serum IL-11 (r = -0.61; P <
0.001). The higher the serum PTH, the lower the IL-11. There was no
correlation between serum calcium concentrations and circulating levels
of IL-11.
IL-6 levels fall and IL-11 levels rise after parathyroid
adenomectomy
To determine whether the level of circulating IL-11 is influenced
by rapid changes in PTH, we measured serum IL-11 and IL-6 every 4
h for 24 h after successful parathyroid adenomectomy in three
patients. As illustrated in Fig. 2, adenomectomy resulted in a rapid decline in circulating IL-6 from a
preoperative value of 21.5 ± 2.7 pg/ml to a value of 7.1 ±
0.6 pg/ml 24 h after surgery, a decline of 67% (P
= 0.03). This was accompanied by a 2-fold rise in mean serum IL-11
levels (mean preoperative value, 4.3 ± 0.7 pg/ml; 24 h
postsurgery, 9.9 ± 1.7; P = 0.03), suggesting a
reciprocal relationship between circulating levels of IL-6 and
IL-11.
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As shown in Fig. 3, PTH infusion resulted
in a progressive increase in circulating IL-6 levels, whereas no change
in IL-6 levels was observed in vehicle-infused animals [end
vs. start of PTH infusion, 20.0 ± 1.6 vs.
5.1 ± 0.6 pg/ml (P < 0.001); vehicle-infused,
4.0 ± 0.1 vs. 3.7 ± 0.3 pg/ml (P
= NS)]. In contrast, PTH infusion caused a time-dependent decline in
circulating IL-11 values such that at the end of the infusion mean
values were significantly below those at the start and lower than those
at the end of the vehicle infusion [end vs. start of PTH
infusion, 5.8 ± 0.2 vs. 8.0 ± 0.4 pg/ml
(P < 0.05); end of PTH infusion vs. end of
vehicle infusion, 5.8 ± 0.2 vs. 7.5 ± 0.3 pg/ml
(P < 0.05)].
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). Thus, at the end of the 5-d infusion,
mean IL-11 levels in the three groups of animals were 4.0 ± 0.2
pg/ml in PTH-infused animals, 6.2 ± 0.3 pg/ml in PTH-infused
animals pretreated with nonimmune serum, and 13.4 ± 0.7 pg/ml in
PTH-infused mice pretreated in neutralizing antiserum to IL-6
(P < 0.0001 compared with the other two groups). The
apparent decline in the circulating IL-6 concentration after treatment
with neutralizing antibody to IL-6 is probably an artifact due to
binding of the antibody near or to an epitope recognized by the
antibody used in the ELISA.
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).
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). As SaOS-2 is a malignant cell
line, these studies were repeated using primary human and murine
osteoblast cultures. Consistent with the findings in the SaOS-2 cells,
PTH induced a 2.1 ± 0.1-fold increase in IL-11 levels in the
presence of nonimmune serum (64 ± 3 to 134 ± 6 pg/ml) and a
3.0 ± 0.1-fold increase in the presence of neutralizing antiserum
to IL-6 (63.7 ± 2.6 to 191.2 ± 5.2 pg/ml) in primary human
osteoblast cultures (P < 0.01 for fold increase
compared with the fold increase seen in the presence of nonimmune
serum). In the primary murine osteoblasts, PTH induced a 2.0 ±
0.1-fold increase in IL-11 levels in the presence of nonimmune serum
(39 ± 3 to 77 ± 4 pg/ml) and a 2.9 ± 0.2-fold
increase in the presence of neutralizing antiserum to IL-6 (39 ±
3 to 115 ± 9 pg/ml; P < 0.01 for the fold
increase compared with the fold increase seen in the presence of
nonimmune serum).
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). In four separate
experiments, PTH-induced IL-11 mRNA expression was not different in the
presence of neutralizing antiserum to IL-6 compared with nonimmune
serum. The data were evaluated in two ways. First, each IL-11
transcript densitometric measurement was corrected for loading by
normalizing to GAPDH transcript expression, and the mean values for the
four experiments were compared under the four experimental conditions.
These values were: nonimmune serum alone, 0.91 ± 0.32;
IL-6-neutralizing antibody alone, 0.75 ± 0.20; PTH plus nonimmune
serum, 2.16 ± 1.27; and PTH plus IL-6-neutralizing antibody,
1.78 ± 0.78 (P = 0.27 for the last two groups).
In the second method we subtracted the values obtained when cells were
treated with only nonimmune serum or anti-IL-6 antibody from the values
in the respective PTH-treated cells. These values were: PTH plus
nonimmune serum, 1.25 ± 1.05-fold; PTH plus anti IL-6 antibody,
1.04 ± 0.60-fold (P = 0.49). These data suggest
that regulation of IL-11 by IL-6 occurs at a posttranscriptional
level.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PTH exerts its proresorptive effects principally by stimulating the production of soluble and cell surface molecules in osteoblasts and stromal cells. Both IL-11 and IL-6 are produced by osteoblasts in response to PTH and are thought to act principally by enhancing the formation of osteoclast precursors. The physiological importance of the inverse relationship between IL-6 and IL-11 in response to PTH may be in modulating the total resorptive effect of PTH in vivo. Thus, a reduction in IL-11 when IL-6 levels are elevated may restrain the resorptive response to PTH by diminishing osteoclast progenitor recruitment.
Alternatively, this coordinate regulation may be part of the control mechanisms for the overlapping biological function of these two cytokines. Thus, for example, in the absence of IL-6 (e.g. IL-6-/- mice) when the resorptive actions of PTH are impaired (14), these animals are only modestly hypocalcemic (23), suggesting that other factors, such as IL-11, compensate in the absence of IL-6 as downstream effectors for PTH’s resorptive action.
The tissue sites at which this coordinate regulation occurs cannot be
identified with certainty from our study. However, our data indicate
that at least one site where this interaction occurs is in osteoblasts.
Although down-regulation of IL-11 by IL-6 has, to our knowledge, not
been previously reported, the converse has been observed. Pretreatment
of peritoneal macrophages with IL-11 resulted in a significant (>60%)
reduction in lipopolysaccharide-induced IL-6 production
(5). In macrophages this inhibition is transcriptionally
regulated by induction of I
B family members that inhibit
transcriptional activation of NFB (24). It is
postulated that this coordinate regulation restrains the resultant
inflammatory response. Thus, IL-11 has been shown to improve survival
due to sepsis in two experimental models (25, 26). In bone
tissue, the inverse relationship described herein, namely suppression
of IL-11 by IL-6, may result in an analogous effect, that is to
restrain the net resorbing effect of PTH. Thus, tight coordinate
regulation of IL-6 and IL-11 expression in both the immune and skeletal
systems may be required to prevent untoward effects on the
organism.
Animals in which the IL-6 or IL-11 genes have been deleted and transgenic animals with targeted overexpression of either cytokine in osteoblasts would be interesting models in which to further explore this regulation. For example, mice overexpressing IL-11 in bone marrow have normal rates of bone resorption (27). In the light of our data, it may be informative to assess the level of IL-6 expression in the serum and marrow of these animals.
Our data may have some relevance to the syndrome of mild primary hyperparathyroidism, where bone loss rates are modest if they occur at all. It may be that down-regulation of IL-11 prevents a more significant skeletal effect of PTH from being manifested. It will be of interest to measure circulating levels of IL-11 in subsets of patients with severe hyperparathyroidism to determine whether this negative regulation is overcome, and IL-11 levels are also increased.
The mechanism by which IL-6 down-regulates IL-11 production appears, at least in bone cells, not to be transcription dependent. Neutralizing antibody to IL-6 did not influence the extent to which PTH increased IL-11 transcript expression. This raises the possibility that IL-6 directly or indirectly influences the translation efficiency of IL-11 mRNA or the rate of IL-11 protein turnover.
In conclusion, we have established that increased IL-6 production in response to PTH results in decreased circulating IL-11 levels due to down-regulation of IL-11 production. At least one tissue in which this occurs is bone. As both IL-6 and IL-11 promote osteoclast formation, down-regulation of IL-11 by IL-6 may be one way in which the total resorptive activity of PTH is modulated.
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Acknowledgments |
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Footnotes |
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Abbreviations: CV, Coefficient of variation; hPTH, human PTH.
Received November 1, 2000.
Accepted for publication May 10, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are elevated in primary hyperparathyroidism and
correlate with markers of bone resorption - a clinical research center
study. J Clin Endocrinol Metab 81:3450–3454[Abstract]
B. J Immunol 159:5661–5670[Abstract]
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,
IL-6; •, IL-11.
, P < 0.001
vs. PTH and nonimmune serum. NIS, Nonimmune serum; anti
IL-6, neutralizing antibody to IL-6.
) or PTH-treated cultures in the
presence of neutralizing antibody to IL-6 (
). * *,
P < 0.001; *, P < 0.01
(compared with treatment with nonimmune serum).