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Section of Clinical Immunology and Infectious Diseases and Research Institute for Internal Medicine, Medical Department A (P.A., F.M., S.S.F.), and the Section of Endocrinology, Medical Department B (T.U., J.B.), Rikshospitalet, N-0027 Oslo; and the Institute of Cancer Research and Molecular Biology, The Norwegian University of Science and Technology (E.L., T.E.), 7005 Trondheim, Norway
Address all correspondence and requests for reprints to: Dr. Pål Aukrust, Section of Clinical Immunology and Infectious Diseases, Medical Department A, Rikshospitalet, N-0027 Oslo, Norway. E-mail: pal.aukrust{at}klinmed.uio.no
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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(TNF) may stimulate osteoclast activity (1, 2, 3), and enhanced IL-6
levels appear to play a pathogenic role in the enhanced bone resorption
of postmenopausal osteoporosis (4, 5). Some of these cytokines
(e.g. IL-1 and TNF) may also inhibit bone formation
exerted by their negative regulatory effects on osteoblasts (1, 6, 7).
This inhibitory effect on osteoblasts combined with stimulation of
osteoclasts suggest a pathogenic role for these cytokines in bone
disorders characterized by increased resorption combined with
decreased formation of bone, e.g. osteomyelitis, rheumatoid
arthritis, and certain malignancies (1).
Persistent activation of proinflammatory cytokines such as IL-1,
and in particular TNF, appears to play an important pathogenic role
in human immunodeficiency virus (HIV) infection (8, 9, 10). This
proinflammatory activation may enhance HIV replication, contribute to
the development of immunodeficiency and certain clinical manifestations
(10, 11), and be related to the endocrine abnormalities seen in
HIV-infected individuals (12, 13). Thus, we have previously
demonstrated a marked decrease in serum levels of 1,25-dihydroxyvitamin
D3 [1,25-(OH)2D] in HIV-infected patients
correlated with the degree of immunodeficiency, possibly related to
enhanced TNF activity (14, 15, 16). Vitamin D metabolites have complex
effects on the bone system, with stimulatory effects on both formation
and resorption (1, 17). Of particular interest, the stimulatory effects
of 1,25-(OH)2D on osteoblast function appear to be
inhibited by TNF (18, 19). Based on the roles of cytokines and
1,25-(OH)2D in bone homeostasis, we examined the
possibility that HIV-infected patients, characterized by enhanced
levels of proinflammatory cytokines and 1,25-(OH)2D
deficiency, may develop disturbed bone metabolism, as evaluated by
serum markers of bone formation and resorption.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Seventy-three HIV-infected patients (58 men and 15 women; median
age, 38 yr; range, 19–58 yr) were included in the study (Table 1
). Clinically, 16 patients were
classified as asymptomatic HIV-infected patients [Centers for Disease
Control and Prevention (CDC) group A], 19 patients were classified as
symptomatic non-AIDS patients (CDC group B), and 38 were classified as
acquired immunodeficiency syndrome (AIDS) patients (CDC group C) (20).
Patients with any of the following criteria were excluded from the
study: recent history of extended bed rest, previous diagnosis of
metabolic bone disorders or endocrine diseases, serum creatinine more
than 100 µmol/L or alanine aminotransferase more than 50 U/L, abuse
of drugs or alcohol, and the use of corticosteroids, diuretics,
cytostatics, anticoagulants, nonsteroid antiinflammatory drugs,
forscarnet, or any other drugs with known effect on bone metabolism. At
the time of the study, none of the patients had any signs or symptoms
of acute or exacerbation of chronic disease. In the cross-sectional
testing (see below), 49 patients received antiretroviral therapy (35
zidovudine, 3 didanosine, and 11 zidovudine
in combination with lamivudine). Forty-nine patients
received Pneumocystis carinii prophylaxis (8 aerosolized
pentamidine, 30 dapsone, and 11 trimetoprim-sulfametoxazole). Sixteen
of the patients were followed during highly active antiretroviral
therapy (HAART), and all of these received indinavir (800 mg, 3 times
daily) in combination with zidovudine (250 mg, twice
daily) and lamivudine (150 mg, twice daily). Controls in
the study were 25 sex- and age-matched HIV-seronegative healthy blood
donors (19 men and 6 women; median age, 39 yr; range, 20–65 yr).
Informed consent for blood sampling was obtained from all patients and
controls. Blood samples for the study were drawn between 0800–1000 h
after an overnight fast as previously described (9). The serum samples
were stored at -70 C until analysis and were thawed only once. All
samples from a given patient were analyzed at the same time to minimize
the run to run variability.
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Osteocalcin levels were measured by an immunoradiometric assay (Instar Corp., Stillwater, MI) detecting intact osteocalcin-(1–49). Degradation products of the C-terminal telopeptides of type I collagen (Crosslaps) were measured by an enzyme immunoassay (EIA; Osteometer Bio Tech, Herlev, Denmark).
Quantification of 1,25-(OH)2D and PTH in serum
1,25-(OH)2D was analyzed by RIA (Nichols Institute Diagnostics, Diagnostics B.V., Wijchen, The Netherlands) (14). Intact PTH was quantified using a solid phase, two-site chemiluminescent enzyme immunometric assay (Diagnostic Products Corp., Los Angeles, CA) (16).
EIA for detection of TNF and soluble TNF receptors (TNFRs)
The soluble TNFRs, p55- and p75-TNFR, were analyzed by EIAs as
previously described (21). TNF was quantified by EIA (Medgenix,
Fleurus, Belgium) (9).
Miscellaneous
Serum levels of ionized calcium and magnesium were analyzed as described previously (16). Plasma (ethylenediamine tetraacetate) HIV ribonucleic acid levels were measured by quantitative reverse PCR (Amplicor HIV Monitor, Roche Diagnostic Systems, Branchburg, NJ; detection limit, 200 copies/mL). CD4+ and CD8+ T cell counts in peripheral blood were determined by immunomagnetic quantification (9).
Statistical analyses
When comparing more than two groups, the Kruskal-Wallis test was used. If a significant difference was found, the Mann-Whitney U test (two-tailed) was used to determine the differences between each pair of groups. Coefficients of correlation were calculated by the Spearman rank test. During HAART, each parameter were compared with baseline by Wilcoxon’s rank sum test for paired data. Data are given as medians and 25–75th percentiles if not otherwise quoted. P values are two-sided and considered significant when <0.05.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We first measured serum levels of osteocalcin and
C-telopeptide as indirect parameters of bone formation and
resorption, respectively. Patients in CDC group C had significantly
decreased osteocalcin levels compared both with healthy controls and
other HIV-infected patients (Fig. 1). In
fact, 11 of the patients, all in CDC group C, had osteocalcin levels
below the detection limit of the assay (0.034 nmol/L) compared with 1
healthy control (Fig. 1). In contrast to the decreased osteocalcin
levels, patients in CDC group C, but not those in CDC groups A and B,
had raised C-telopeptide levels with more than a 2-fold increase
compared with controls in 10 of the patients, representing those AIDS
patients with the most advanced disease. The abnormalities in serum
concentrations of osteocalcin and C-telopeptide were also found when
males and females were examined separately (data not shown). Among
HIV-infected patients, there was no correlation between osteocalcin and
C-telopeptide levels (r = -0.06; P = 0.62).
Although C-telopeptide was negatively correlated with CD4+
T cell counts (r = -0.28; P < 0.02), osteocalcin
levels were positively correlated (r = 0.46; P <
0.001) with numbers of this lymphocyte subset in the patient group.
Osteocalcin levels were also positively correlated with
CD8+ T cell counts (r = 0.28; P <
0.03) and negatively correlated with plasma viral load (r =
-0.33; P < 0.03).
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HIV-infected patients had markedly elevated serum levels of TNF
and both types of soluble TNFRs, with particularly high levels in CDC
group C (Table 1
). Interestingly, there was a strong inverse
correlation between levels of both p55-TNFR and p75-TNFR and
osteocalcin (Fig. 2). Also, TNF tended
to be inversely correlated with osteocalcin levels (r = -0.24;
P = 0.06). In contrast to the negative correlation with
osteocalcin, p75-TNFR were positively correlated with C-telopeptide
(Fig. 2). Notably, also within the AIDS group, but not within CDC
groups A and B, C-telopeptide was significantly correlated with soluble
p75-TNFR (r = 0.50; P = 0.002), reflecting that
the increase in C-telopeptide concentrations was mostly restricted to
AIDS patients with the most marked activation of the TNF system.
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As previously reported by us (14, 15, 16), AIDS patients had a
profound decrease in serum levels of 1,25-(OH)2D associated
with decreased PTH and normal or decreased calcium and magnesium levels
(Table 1). However, there was no significant correlation between
1,25-(OH)2D, calcium, magnesium, or PTH levels and neither
osteocalcin nor C-telopeptide levels (data not shown).
Parameters of bone remodeling during HAART
To further elucidate the interaction among HIV, TNF, and bone
remodeling, we studied the effect of HAART on serum levels of
osteocalcin and C-telopeptide in 16 HIV-infected patients who were
followed for 24 months. At baseline, C-telopeptide levels were
different from the corresponding levels in the AIDS group presented in
the cross-sectional study (Fig. 1). However, only 5 of the 16 patients
receiving HAART were classified in the AIDS group, and most of these
had less advanced disease than the AIDS patients with elevated
C-telopeptide levels described in Fig. 1. After initiating therapy,
there was a marked decrease in viral load [maximal decrease in HIV
ribonucleic acid copies/mL: 2.29 (range, 1.43–2.67) log10;
P < 0.01] and levels of TNF components
(P < 0.01), and a marked increase in CD4+
T cell counts [maximal increase, 110 (range, 60–155) x
106/L; P < 0.01]. Concomitantly, there
was a significant and prolonged increase in osteocalcin levels (Fig. 3). In fact, HAART induced a rise in
osteocalcin levels from concentrations below the normal range at
baseline to levels above those in healthy controls at the end of the
study (Fig. 3). Also, serum levels of C-telopeptide (Fig. 3) and PTH
(data not shown) tended to increase in the second part of the study
period, but this increase did not reach statistical significance (Fig. 3). Notably, although there was no correlation between serum levels of
osteocalcin and C-telopeptide at baseline, after HAART these parameters
were significantly correlated when comparing the last serum samples
from each patient (Fig. 4).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Skeletal integrity is maintained by a dynamic process of cellular events, with osteoclastic bone resorption and simultaneous formation of new bone by osteoblasts (1). During normal bone turnover these processes are closely regulated and synchronized. Although osteocalcin levels may not necessarily reflect osteoblast function (1), our findings of markedly decreased osteocalcin and increased C-telopeptide levels during advanced HIV-related disease suggest that there is a disturbance in this synchronized bone-remodeling process in these patients.
We found that the disturbances in bone metabolism during HIV infection
were correlated with increased activity of the TNF system. This finding
may reflect important pathogenic mechanisms in bone remodeling in HIV
infection. Although several cytokines, such as IL-1, IL-6, IL-11, and
TNF, may stimulate osteoclast activity, some of these cytokines, in
particular IL-1 and TNF, may also inhibit osteoblast function
(1, 2, 3). Low PTH levels, as found in HIV-infected patients, may also
impair bone formation (24), but we found no correlation between PTH and
osteocalcin levels. Decreased insulin-like growth factor (25) and
increased corticosteroid (26) levels have been found in HIV-infected
patients, and these factors may affect bone remodeling (1). Direct
HIV-mediated effects on osteoblasts or precursors of osteoclasts may
also play a role (27, 28, 29). However, although several factors probably
are involved, our results suggest that increased activity of TNF
and/or cytokines induced by TNF (e.g. IL-1 and IL-6) may
play an important role in mediating disturbed bone metabolism during
HIV infection. Of particular interest is the recently identified
osteoprotegerin and its ligand, which are new members of the TNFR/TNF
family and regulate osteoclast differentiation and activation (30). One
possibility could be that HIV-infected patients may also have increased
levels of the TNF-related molecule osteoprotegerin ligand, which, in
turn, may activate osteoclasts in these patients (30).
As previously reported by us (14, 15, 16), HIV-infected patients with
advanced clinical disease are characterized by markedly decreased serum
levels of 1,25-(OH)2D combined with decreased PTH and
normal or decreased calcium levels, possibly reflecting inhibitory
effects of TNF on 1-hydroxylase activity and PTH secretion (15, 16). Although there were no statistical correlations between
1,25-(OH)2D and either osteocalcin or C-telopeptide levels,
the 1,25-(OH)2D deficiency may be of importance for the
abnormalities in these markers of bone remodeling in HIV infection.
1,25-(OH)2D deficiency may impair the proliferation of
osteoblasts and their precursors at different stages of differentiation
(1). Notably, the effect of 1,25-(OH)2D on osteoblasts
appears to be inhibited by TNF (18, 19). Thus, it is possible that
low 1,25-(OH)2D levels may have a synergistic effect with
increased TNF activity in the induction of disturbed bone homeostasis
during HIV infection.
An interesting finding in the present study was the major alterations
in markers of bone remodeling induced by HAART. Thus, the profound
effect of such therapy on viral load, CD4+ T cells, and TNF
components was associated with a marked rise in serum osteolcalcin
levels from concentrations below to levels above those in controls.
Furthermore, there was a shift from no to a highly significant
correlation between osteocalcin and C-telopeptide, suggesting
synchronization of bone remodeling and enhanced bone turnover during
HAART. The reasons for these changes in markers of bone remodeling
during therapy are uncertain. Although inhibition of direct
HIV-mediated effects on osteoblasts and osteoclasts may be involved, it
is tempting to hypothesize that these findings at least partly reflect
down-regulated activity of TNF and/or other proinflammatory cytokine
systems. It has been suggested that therapy inhibiting activation of
the transcriptional factor nuclear factor-
B, a potent inducer of
several proinflammatory cytokines, may hold therapeutic potential in
the treatment of osteoporosis and other bone disorders (31). The
present study, suggesting synchronization of bone remodeling along with
immunomodulating effects of HAART, may further support such an idea.
However, to determine whether the changes in biochemical parameters of
bone metabolism during HAART are beneficial, larger long term studies
that also include bone density tests will have to be performed.
Decreased bone mineral density has previously been reported in HIV-infected patients (23), and although the clinical consequences of our findings with disturbed osteocalcin and C-telopeptide levels remain unclear, it is conceivable that if these abnormalities persist over time, they may well lead to clinically significant bone disease. Moreover, our findings suggesting synchronization of bone formation and resorption during HAART may represent a previously unrecognized beneficial effect of such therapy, expanding our knowledge of the interactions between cytokines and bone in the bone-remodeling process.
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Acknowledgments |
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Footnotes |
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Received August 11, 1998.
Revised September 30, 1998.
Accepted October 13, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. Inflammation. 18:267–284.[CrossRef][Medline]
and soluble TNF receptors in human immunodeficiency virus type
1 infection–correlations to clinical, immunologic, and virologic
parameters. J Infect Dis. 169:420–424.[Medline]
decreases 1,25-dihydroxyvitamin
D3 receptors in osteoblastic ROS 17/2.8 cells. J Bone
Miner Res. 8:997–1003.[Medline]
in the rat osteocalcin
gene. Endocrinology. 134:1113–1120.B and bone: the
breaking point. Nat. Med. 3:189–190.
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