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Published ahead of print on May 4, 2006, doi:10.1164/rccm.200512-1943OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 306-311, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200512-1943OC

Original Article

Osteoclastogenesis during Infective Exacerbations in Patients with Cystic Fibrosis

Elizabeth F. Shead, Charles S. Haworth, Elaine Gunn, Diana Bilton, Mike A. Scott and Juliet E. Compston

Department of Haematology, NHS Foundation Trust, Addenbrooke's Hospital; Adult Cystic Fibrosis Centre, NHS Foundation Trust, Papworth Hospital; Department of Medicine, University of Cambridge, Cambridge; and Department of Anatomy, University of Bristol, Bristol, United Kingdom

Correspondence and requests for reprints should be addressed to Elizabeth F. Shead, B.Sc., Box 234, Haematology Department, Addenbrooke's Hospital, NHS Foundation Trust, Hills Road, Cambridge CB2 2QQ, UK. E-mail: lizzshead{at}hotmail.com


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Adults with cystic fibrosis (CF) are at increased risk of developing osteoporosis. During infective exacerbations, increased production of proinflammatory cytokines and markers of bone resorption have been reported.

Objective: The aim of this study is to investigate the growth and proliferation of potential osteoclast precursor cells before, during, and after intravenous antibiotic treatment of infective exacerbations in patients with CF.

Methods: Hematopoietic precursor cell growth was examined using colony formation assays using Methocult culture medium. Circulating potential osteoclast precursors were identified using four-color flow cytometry by CD14, CD33, CD34, and CD45 expression.

Results: At the start of an infective exacerbation increases in hematopoietic precursor colony formation (15.42 colonies/105 cells plated, p = 0.025), proliferation (28.5%, p < 0.001), and the numbers of circulating potential osteoclast precursors (6.5%, p < 0.001) were seen in comparison with baseline levels. These increases declined after treatment with intravenous antibiotics to a level close to baseline.

Conclusions: The results demonstrate an increase in the production of potential osteoclast precursors in the peripheral blood during CF infective exacerbations. This may result in increased bone resorption and contribute to bone loss in patients with CF.

Key Words: cystic fibrosis • cytokines • osteoclasts • osteoporosis

Cystic fibrosis (CF) is a disease associated with an increased risk of osteoporosis (1–3) and fracture (4). Previous research suggests a multifactorial etiology, including vitamin D insufficiency, malnutrition, glucocorticoid use, reduced levels of physical activity, and hypogonadism (5). The most consistent correlate of low bone mass, however, is the severity of CF disease, as defined by lung function and nutritional parameters (6). The mechanism of this association remains only partially identified, but may relate to the effects of the systemic inflammatory response to pulmonary infection on osteoclast function (7, 8).

Osteoclasts are large multinucleate cells derived from pluripotent hematopoietic stem cells that separate from the monocyte-macrophage precursors after the colony-forming unit–granulocyte-monocyte (CFU-GM) stage of hematopoiesis (9). CFU-GMs produce cells of the granulocytic and monocytic lineages, which include osteoclast precursors (10). Cells originating from these colonies within the bone marrow develop lineage commitment while circulating in the peripheral blood (11–13), and osteoclasts and precursors can be identified by a number of specific markers including vitronectin receptor, calcitonin receptor, and tartrate-resistant acid phosphatase. Circulating osteoclast precursors then migrate to the bone surface where they mature and become capable of resorbing bone. Osteoclastic activity and development are regulated by other cell types within the bone marrow, including osteoblasts and stromal cells, and also by other factors, such as hormones, cytokines, and growth factors (14).

There is evidence that lung function, intravenous antibiotic requirement, and markers of systemic inflammation, such as C-reactive protein (CRP), are closely linked to the severity of bone disease in patients with CF (1–3). Furthermore, there is some evidence that levels of both circulating inflammatory cytokines and biochemical markers of bone resorption are elevated during exacerbations of pulmonary disease, suggesting that systemic inflammatory mediators affect osteoclast activity, and thereby have a pathogenic role in the development of CF-related osteoporosis (15). The production of interleukin (IL)-1beta and IL-6 increases during infective exacerbations (16). IL-1beta is a cytokine that induces development of osteoclast precursors and increases osteoclast activity, whereas IL-6 plays an important role in regulating osteoclast formation from precursor cells (17). Both these cytokines have been shown to play a role in the increased bone resorption seen in Paget's disease, rheumatoid arthritis (18), and postmenopausal osteoporosis (17). Other cytokines that influence osteoclast activity and survival and are implicated in the pathogenesis of bone disease include tumor necrosis factor {alpha} (19), levels of which are increased in patients with CF during infective exacerbations (7, 8), and receptor activator of nuclear factor (NF)-{kappa}B ligand (20), which has not been studied in the CF population.

The aim of this study is to investigate changes in osteoclastogenesis during infective exacerbations treated with intravenous antibiotics in patients with CF. Some of the results of these studies have been previously reported in the form of conference abstracts (21–23).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Patients were recruited consecutively from the Papworth Hospital Adult Cystic Fibrosis Centre, Cambridge, United Kingdom, using the following inclusion criteria: CF confirmed by gene analysis and abnormal sweat test, age of 18 yr or older, FEV1 less than 85% predicted, more than one course of intravenous (IV) antibiotic therapy for an exacerbation in the preceding year, and primarily colonized with Pseudomonas aeruginosa. Patients were excluded if they had received oral glucocorticoids in the 3 mo before recruitment or during the study, had received bisphosphonates, were pregnant during the study, had renal dysfunction, or had undergone solid organ transplantation.

Fifteen patients (eight men, seven women) were recruited with a mean age (± SD) of 24.2 (5.49) yr. Of the 15 patients all had pancreatic insufficiency, 12 were {Delta}F508 homozygous, one {Delta}F508/Q220X, and two {Delta}F508/–. Baseline mean FEV1 was 49.9% (19); mean body mass index, 20.8 (2.6) kg/m2; and mean time since last infective exacerbation was 85 (31) d and last exacerbation requiring IV therapy was 98 (44) d (Table 1). Two patients had received oral prednisolone in the 12 mo before recruitment. Six healthy control subjects were recruited from Addenbrooke's NHS Trust staff (three men, three women) with a mean age of 23.3 (1.21) yr. The study was approved by the local ethics committee, and informed, written consent was obtained from all patients.


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  		TABLE 1. PATIENT DEMOGRAPHICS AT BASELINE

 
Blood samples were taken at four time points: (1) baseline (patient clinically stable), (2) Day 1 (start of infective exacerbation [24]) and immediately before starting IV therapy, (3) Day 14 (end of IV therapy), and (4) Day 42 (follow-up 4 wk after completing IV therapy).

Mononuclear Cell Separation
Each sample was layered over an equal amount of Lymphoprep (Axis-Shield, Kimbolton, UK) and centrifuged at 1,500 rpm for 30 min. The mononuclear cell layer was removed and washed.

Colony-forming Assays
Mononuclear cells were plated at 3 x 105 cells/ml in Methocult H4534 (Stem Cell Technologies Ltd., London, UK). Cultures were set up in duplicate and incubated at 37°C, 5% CO2–air for 14 d, and colonies counted before harvesting cells into phosphate-buffered saline (PBS). Reproducibility of assays was determined by repeated assays of control cultures.

Flow Cytometry
Whole blood was incubated with combinations of fluorescent antibodies to cell surface markers CD14, CD33, CD34, and CD45 (Dako Cytomation Ltd., Ely, UK) and IgG control antibodies. After incubation and red cell lysis, samples were analyzed using a Beckman Coulter EPICS-XL flow cytometer (Beckman Coulter, High Wycombe, UK). Size of the CD14/33/45-positive CD34-negative population (OPP) was measured as a percentage of the total cell count.

DNA Analysis
Harvested colonies were washed in PBS and cell DNA content measured using propidium iodide DNA staining (Beckman Coulter) following the manufacturer's protocol. DNA content was measured using a Beckman Coulter EPICS-XL and the number of cells containing more than one set of chromosomes expressed as a percentage of total cell number (percentage proliferation).

Statistical Analysis
Baseline measurements were compared with those of healthy control subjects using a Mann-Whitney test. Time points were compared using Wilcoxon signed rank test for paired data and the p value adjusted for multiple testing using the Bonferroni correction. For all tests p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
All results are expressed as median with interquartile range (IQR; 25–75%). Box and whisker plots represent median, IQR, and upper and lower extremes of the dataset.

Hematopoietic Colony Growth Increases during Infective Exacerbations
Results showed that the repeatability for CFU-GM colony assays using a cryopreserved control sample was not significantly different (p > 0.05) over five repeats set up at weekly intervals (CV = 22.8%).

A significant increase in CFU-GM colony number from baseline (median, 11.2 colonies/105 cells plated; IQR, 8–18.7) was observed at Day 1 (median, 15.42 colonies/105 cells plated; IQR, 8.6–24.4; p = 0.025; Figure 1). No significant difference was seen between Day 14 (median, 13.4 colonies/105 cells plated; IQR, 10.4–20.4) and baseline values. A subsequent decrease compared with Day 1 was seen by Day 42 (median, 11.5 colonies/105 cells plated; IQR, 7.5–16; p < 0.01). CFU-GM numbers at baseline were not significantly different to those seen at Day 42 (p = 0.19). CFU-GM colony formation at baseline in patients did not differ significantly from that found within the control group (median, 13.5 colonies/105 cells plated; IQR, 8.5–17.5; n = 6).


Figure 1
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  		Figure 1. Colony-forming unit–granulocyte-monocyte (CFU-GM) colony numbers at each time point (n = 15) after 14 d in culture, showing a significant increase in formation at Day 1 (*p = 0.025) compared with baseline, with a subsequent decrease from this peak (at Day 1) by Day 42 (**p < 0.01).

 
To demonstrate the osteoclastic potential of CFU-GM cells, cells from harvested colonies were plated at 3 x 105 cells onto Osteologic slides (BD Biosciences, Oxford, UK). In the presence of receptor activator of NF-{kappa}B ligand and macrophage colony–stimulating factor, the cells produced mature resorbing osteoclasts (Figure 2).


Figure 2
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  		Figure 2. Multinucleate osteoclast (arrow) on an Osteologic slide coated with calcium phosphate formed from CFU-GM colony cells, stained with Diff-Quick. Original magnification x200.

 
All CFU-GM colonies were identified microscopically using identification criteria provided by Stem Cell Technologies (Figure 3).


Figure 3
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  		Figure 3. CFU-GM colony (x100 magnification) showing characteristics used for identification; a flat colony consisting of translucent small and large cells (> 20–50 cells/colony).

 
Increased CFU-GM Proliferation at Times of Infective Exacerbation
Compared with baseline, significant increases (p < 0.001) in CFU-GM cell proliferation were seen at Day 1 (median, 28.5%; IQR, 27.25–34.65) with a subsequent significant decrease (p < 0.01) by Day 14 (median, 18.6%; IQR, 16.9–22.9). By Day 42, proliferation rate had increased slightly to a level comparable with baseline (median, 25.2%; IQR, 22.4–27.4; Figure 4).


Figure 4
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  		Figure 4. Proliferation (percentage of dividing cells) at each time point (n = 15) as measured by propidium iodide DNA staining. Data show a significant increase at Day 1 (**p < 0.001) compared with baseline and a decrease after intravenous antibiotic therapy by Day 14 (*p < 0.01) from Day 1.

 
No correlation between CFU-GM colony numbers and proliferation rate (nonparametric Spearman rank r2 = –0.047, p = 0.81 two-tailed) was seen at any time point. Levels of cell proliferation at baseline did not differ significantly from those found within the control group (median, 26.2%; IQR, 23.4–28.5; n = 6).

OPP Numbers Increase during Infective Exacerbations
A significant increase in the OPP was seen at Day 1 (median, 6.5%; IQR, 6.2–7.1; p < 0.001) and a nonsignificant increase by Day 14 (median, 6.5%; IQR, 4.9–7.3; p = 0.06), compared with baseline (median, 5.3%; IQR, 4.4–6). By Day 42, a decrease (median, 4.9%; IQR, 4.3–6.6) was seen in population size, significantly lower (p < 0.001) than levels at Day 1. OPP numbers at Day 42 were significantly lower than those at baseline (p < 0.05). OPP numbers at baseline did not differ significantly from those found in the normal control subjects (median, 5.6%; IQR, 4.7–6.4; n = 6, Figure 5).


Figure 5
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  		Figure 5. Circulating potential osteoclast precursors throughout an infective exacerbation (n = 15). Flow cytometric analysis of the circulating CD45/CD33/CD14+ CD34– population shows a significant increase at Day 1, compared with baseline (*p < 0.001), decreasing after intravenous antibiotic therapy (Day 42) compared with Day 1 (*p < 0.001) and baseline (**p < 0.05).

 
Moderate increases in white cell count (including monocyte count) were seen at the start of an exacerbation, with the count returning to baseline values after intravenous antibiotic therapy. Total white cell count was not correlated with OPP, although monocyte count was strongly correlated (nonparametric Spearman rank r2 = 0.447, p = 0.03 two-tailed).

No correlation was seen between the size of the OPP and CFU-GM proliferation rate (Spearman rank r2 = 0.02, p = 0.91 two-tailed). A significant correlation was seen, however, between the OPP and CFU-GM formation (nonparametric Spearman rank r2 = 0.17, p = 0.003 two-tailed).

Measures of Inflammatory Status
Levels of CRP varied (not significant) during an infective exacerbation with a trend for increase at Day 1 (median, 15 mg/L; IQR, 7–67.5) compared with baseline (median, 11 mg/L; IQR, 6.5–23.5) and a subsequent fall by Day 42 (median, 8 mg/L; IQR, 6–17; Figure 6). This trend (increase at Day 1, fall by Day 42) mimics the trend seen in OPP size and CFU-GM number. CRP levels were not correlated with OPP (nonparametric Spearman rank r2 = 0.13, p = 0.46), CFU-GM number (nonparametric Spearman rank r2 = 0.17, p = 0.25), or CFU-GM proliferation rate (nonparametric Spearman rank r2 = 0.28, p = 0.07).


Figure 6
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  		Figure 6. C-reactive protein (CRP) levels at each time point (n = 15). Levels of CRP vary over the time points with a trend for an increase at Day 1 and a decrease by Day 42.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data show that the potential to form osteoclasts from hematopoietic precursors is increased at times of infective exacerbation in adults with CF, as demonstrated by an increase in CFU-GM growth and proliferation and increased numbers of the circulating OPP. Further evidence is provided by the positive correlation between the OPP and number of circulating monocytes, from which the osteoclast precursors develop. The mechanisms responsible have not been clearly defined but may be related to increased levels of circulating inflammatory markers as reported by others (25). Increased osteoclast formation at times of exacerbation may contribute to bone loss and the increased risk of osteoporosis in these patients. In this study, infective exacerbations were defined using criteria provided by Fuchs and coworkers (24). Our data have also shown that the numbers of circulating potential osteoclast precursors decrease with IV antibiotic therapy.

The pathophysiology of CF-associated osteoporosis has been investigated using bone histomorphometric analysis. Elkin and coworkers (26) studied biopsies from clinically stable patients and reported that the predominant change was that of low bone turnover and reduced bone formation at the cellular level. There was considerable heterogeneity among the patients, however, and in some there was evidence of increased bone resorption. In contrast, a study of autopsy specimens demonstrated that increased bone resorption was the most common finding (27), but the source of the specimens indicates that the patients had end-stage disease and most were immunosuppressed, having undergone lung transplantation. Together, these studies are consistent with the hypothesis that CF-related low bone mineral density results from a combination of low bone turnover and formation during periods of disease stability, with episodes of increased bone turnover and resorption during infective exacerbations.

The effects of infective exacerbation on bone metabolism in patients with CF have been reported in three studies using serum levels of cytokines and biochemical markers of bone turnover. Aris and coworkers (7) and Ionescu and coworkers (8) demonstrated decreases in levels of N-telopeptide, a biochemical marker of bone resorption, after successful antibiotic therapy, implying a reduction in bone resorption. In addition, Aris and coworkers (7) reported increased levels of osteocalcin (a marker of bone formation) after therapy and decreased levels of CRP, a marker of inflammatory status. Serum levels of IL-6 and tumor necrosis factor {alpha} had also decreased by the end of therapy, and in the study of Ionescu and coworkers (8), an inverse relationship between levels of these cytokines and bone mineral density was demonstrated. Furthermore, Haworth and coworkers (25) in a prospective study reported a significant correlation between mean levels of IL-6 and biochemical markers of bone resorption, levels of IL-6 being independent predictors of bone loss over 1 yr. In the present study, we did not find a significant correlation between CRP levels and OPP, possibly as a result of the relatively small sample size. Collectively, these data suggest that inflammation may play a role in the loss of bone mineral density and subsequent development of osteoporosis in patients with CF.

An association between inflammation and bone disease is well recognized and emphasizes the importance of mediators of the immune response as regulators of bone remodeling (28). One example is rheumatoid arthritis, a systemic autoimmune disease in which there is stimulation of the immune system and joint destruction, the latter being attributed to increased osteoclast activity (29). Subsequent work has shown a specific role for T cells in this disease-associated bone destruction (30). Other diseases in which inflammation may result in bone loss are inflammatory bowel disease (31) and chronic obstructive pulmonary disease (32). The data in these studies further support the hypothesis that systemic inflammation stimulates the formation of osteoclasts and may influence the development of bone disease. The systemic inflammatory response has also been shown to be associated with reductions in fat-free mass in patients with CF and chronic obstructive pulmonary disease (8, 32), which in turn may contribute to reduced bone mineral density.

Our study has two main limitations. First, the number of patients studied was relatively small and the statistical power to demonstrate changes was limited. Because similar studies of OPP have not been previously reported, it was not possible to perform power calculations before the study. Second, measurements of serum cytokine levels and biochemical markers were not included in this study, and thus we were unable to document whether the increase in osteoclastogenesis was associated with increased systemic cytokine levels and biochemical evidence of increased bone resorption.

This study supports the hypothesis that systemic inflammation may contribute to bone loss in CF and we speculate that during infective exacerbations the inflammatory response may result in increased formation of osteoclasts, contributing to the increased bone resorption seen in adults with CF, if the osteoclast precursor population were to become active (7, 8, 25). The mechanism we have suggested may not be specific to CF and may play a role in the pathogenesis of osteoporosis in other inflammatory conditions. Increased osteoclast precursor numbers have been reported in patients with multiple myeloma (33), although in other inflammatory conditions, such as rheumatoid arthritis, bone loss has been associated with increased osteoclast activity rather than increased number of precursors (29, 34). Disease severity is the most consistent correlate of reduced bone mineral density (1–3) in patients with CF, suggesting that the increased bone turnover during infective exacerbation has a cumulative effect over time and increases the risk of osteoporosis. Finally, further research is required to characterize mechanisms by which the formation of osteoclast precursors is stimulated during infective exacerbations and whether these precursors become fully functioning mature osteoclasts during this time.


   Acknowledgments
 
The authors thank all the members of the Bone Research Group, University of Cambridge, and all the staff within the Haematology Department, Addenbrooke's Hospital, NHS Foundation Trust, and the Adult Cystic Fibrosis Centre, Papworth Hospital, NHS Foundation Trust, for their help and guidance with this study. They also thank Linda Sharples, MRC Biostatistics Unit, Cambridge, for statistical advice relating to this work.


   FOOTNOTES
 
Supported by an NHS trainee clinical scientist (hematology) program funded by the Workforce Development Confederation.

Originally Published in Press as DOI: 10.1164/rccm.200512-1943OC on May 4, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form December 21, 2005; accepted in final form May 3, 2006


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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