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Original Studies |
Section of Clinical Immunology and Infectious Diseases, Medical Department A, and Research Institute for Internal Medicine, University of Oslo (C.J.H., P.A., F.M., S.S.F.), and the Department of Clinical Chemistry (L.M.), The National Hospital-Rikshospitalet; and the Hormone Laboratory, Aker University Hospital (E.H.), Oslo, Norway
Address all correspondence and requests for reprints to: P
l Aukrust, M.D., Ph.D., Research Institute for Internal Medicine, The National Hospital-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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(TNF).
We conclude that inadequate 1-hydroxylation of 25-hydroxyvitamin
D seems to be the most likely cause of 1,25-(OH)2D
deficiency in HIV-infected patients, possibly induced by an inhibitory
effect of TNF. The low 1,25-(OH)2D and high TNF
levels observed may impair the immune response in HIV-infected patients
both independently and in combination and may represent an important
feature of the pathogenesis of HIV-related immunodeficiency. Markedly
depressed 1,25-(OH)2D serum levels are also present in
certain other disorders characterized by immunological hyperactivity.
Thus, the findings in the present study may not only represent a
previously unrecognized immune-mediated mechanism for induction of
1,25-(OH)2D deficiency in human disease, but may also
reflect the importance of adequate serum levels of
1,25-(OH)2D for satisfactory performance of the immune
system in man.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxylation of 25D,
such as hereditary defects of this enzyme (vitamin D-dependent rickets
type I) and severe renal failure (3, 4, 5), leading to disturbances in
calcium and bone metabolism. Until the advent of antibiotics, vitamin D was used to treat mycobacterial infections (6, 7), and vitamin D deficiency has been associated with impaired cellular immunity (8, 9). More recently, a substantial amount of experimental data from in vitro studies has established 1,25-(OH)2D as a potent modulator of cells in the immune system (2, 10, 11).
We have previously, for the first time, reported a marked decrease in serum levels of 1,25-(OH)2D in human immunodeficiency virus (HIV)-infected patients correlating with the degree of immunodeficiency and survival. Particularly low levels were seen in acquired immunodeficiency syndrome (AIDS) patients with ongoing Mycobacterium avium complex (MAC) infection (12, 13). The low 1,25-(OH)2D levels appeared not to be related to deficiency of 25D, and the reason for the low levels in HIV-infected patients remains unclear.
The present study was designed to further examine the reasons for and the metabolic consequences of the low 1,25-(OH)2D serum levels in HIV-infected patients. As 1,25-(OH)2D has a central role in the complex interactions of calcium, phosphate, PTH, and calcitonin in calcium homeostasis, we wanted, in particular, to examine the consequences of severe 1,25-(OH)2D deficiency on these parameters.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Fifty-four consecutively recruited HIV-infected patients (44 males and 10 females; median age, 36 yr; range, 16–64 yr) were included in the study and were classified according to the revised criteria from Centers for Disease Control and Prevention (CDC; 1992) as asymptomatic HIV infection (CDC group A; n = 15), symptomatic non-AIDS HIV infection (CDC group B; n = 12), and AIDS (CDC group C; n = 27). Within a time span of 4 weeks (from 3 weeks before to 1 week after blood sampling), 16 patients had an ongoing clinical event (chronic symptomatic cytomegalovirus-infection, n = 4; chronic MAC infection, n = 3; chronic symptomatic hepatitis C virus infection, n = 1; Candida esophagitis, n = 1; Kaposi’s sarcoma with visceral involvement, n = 2; bacterial pneumonia, n = 3; AIDS dementia complex, n = 1; cryptococcal meningitis, n = 1). Twenty patients had diarrhea (>2 loose stools/day) persisting more than 30 days and weight loss of more than 10% of body weight or both; 4 of these patients were classified as having wasting syndrome (CDC, 1992).
None of the patients was abusing drugs or alcohol at the time of the study, and all except four had serum creatinine levels within the normal range.
Controls in the study were 20 sex- and age-matched healthy blood donors
for tumor necrosis factor- (TNF), and CD4 and CD8 lymphocytes.
For the other parameters, reference values in the laboratory were
used.
Blood-sampling protocol
Blood samples were drawn between 0800–1000h after an overnight fast into sterile vacuum blood collection tubes without any additives. The tubes were immediately immersed in ice water and allowed to clot for less than 1 h before centrifugation. The serum samples were stored at -70 C until analysis and were frozen and thawed only once.
Quantification of serum levels of vitamin D metabolites
Serum levels of 1,25-(OH)2D and 25D were analyzed by two different methods. First, 34 samples were analyzed with RIAs obtained from Nichols Institute Diagnostics (Wijchen, The Netherlands), as described previously (12). Then, 24 samples were analyzed using methods established at The Hormone Laboratory, Aker University Hospital (Oslo, Norway) (14). Briefly, vitamin D3 metabolites were measured after extraction of serum with diethyl ether and chromatographic separation. As quantification of 1,25-(OH)2D requires 2 mL serum, only four samples were analyzed at both laboratories, two with undetectable 1,25-(OH)2D levels and two with serum levels in the normal range. Samples with undetectable serum levels were undetectable in both laboratories; the differences between the other two samples were 11% and 18%, respectively. Samples from the two laboratories were first analyzed separately to look for correlations with clinical and laboratory parameters. When they showed similar patterns, the results from the two laboratories were pooled for further statistical analyses.
Serum levels of vitamin D-binding protein (DBP) was measured by standard methods using rocket immunoelectrophoresis (15).
Quantification of serum levels of PTH
Serum levels of intact PTH were quantified using a solid phase, two-site chemiluminescent enzyme immunometric assay for use with the IMMULITE automated analyzer (Diagnostic Product Corp., Los Angeles, CA).
Quantification of other biochemical parameters
Serum levels of creatinine, albumin, iron, phosphate, and magnesium were analyzed in a Hitachi-917 autoanalyzer, and serum levels of ionized calcium were analyzed in a Hitachi-987 autoanalyzer (Hitachi Scientific Instruments, Tokyo, Japan), both with reagents from Boehringer Mannheim (Mannheim, Germany). Serum levels of prealbumin were quantified by nephelometry calibrated by commercial standards (Boehringer Mannheim). Serum cobalamin and folate in serum and erythrocytes were quantified by standard methods using RIAs from Diagnostic Products Corp. Serum levels of zinc were measured by atomic absorption spectrophotometry (Perkin-Elmer, Norwalk, CT), and calcitonin was determined by RIAs from CIS-Bio International (Gif-sur-Yvette, France).
Quantification of TNF
TNF in serum samples was determined by an enzyme immunoassay
from Medgenix (Fleurus, Belgium) as previously described (13).
Determination of T lymphocyte subsets
The numbers of CD4+ and CD8+ lymphocytes in peripheral blood were determined by immunomagnetic quantification as described previously (12).
Statistical analysis
For comparison of two groups, Fisher’s exact test was used when analyzing table frequencies, a two-tailed Mann-Whitney U test was used when dealing with continuous data, and the Kruskal-Wallis ANOVA was used for comparison of three or more groups. Coefficients of correlation (r) were calculated by the Spearman rank test. For several of the clinical chemistry analyses no control group was available. Instead, a Wilcoxon test for one sample (signed rank sum) was used for the difference di = Xi (variable in clinical group) - m (median of the reference interval). When dealing with symmetrical distributions, m was calculated as the arithmetic mean of lower and upper reference intervals. For PTH and S- and E-folate, the mean was calculated for log-transformed data, and m was the corresponding retransformed value. The same procedure was applied to the calculation of quartiles. Data are given as the median and 25–75th percentiles unless otherwise stated.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In accordance with our previous results, we found that
HIV-infected patients had significantly depressed serum levels of
1,25-(OH)2D compared to controls (Table 1
). Twenty-nine patients had serum levels
below the normal range, and 18 of these had undetectable serum levels
of 1,25-(OH)2D. This marked decrease in serum levels of
1,25-(OH)2D was confirmed by two different methods for
analyzing 1,25-(OH)2D levels (see Materials and
Methods).
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).
When patients with undetectable 1,25-(OH)2D levels were
compared to other patients, there were no significant differences in
levels of either 25D or DBP, although there was a slight reduction in
DBP in patients with undetectable 1,25-(OH)2D levels (Fig. 1, C and D). However, even in these patients DBP levels were within the
normal range.
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Twenty of the patients suffered weight loss and/or diarrhea, but
the 1,25-(OH)2D levels in these patients were not
significantly different from those in other HIV-infected patients (data
not shown). The patients were screened for malabsorption and
malnutrition by analysis of serum levels of albumin, prealbumin, iron,
magnesium, zinc, folate, and vitamin B12 and folate levels in
erythrocytes. Some of these variables were significantly decreased in
the patient group compared with control values (Table 1). However,
there were no correlations between parameters of malabsorption and
malnutrition and 1,25-(OH)2D levels among HIV-infected
patients, and we found no significant differences in these parameters
when patients with undetectable and detectable 1,25-(OH)2D
levels were compared (data not shown)
All four patients diagnosed as having wasting syndrome had undetectable 1,25-(OH)2D levels.
Serum levels of calcium, phosphate, PTH, and calcitonin
As shown in Table 1, serum levels of ionized calcium and PTH were
modestly, but significantly, decreased in HIV-infected patients
compared to controls. However, there were no significant differences in
calcium and PTH levels when patients with undetectable
1,25-(OH)2D levels were compared to other patients (Fig. 1, A and E). There was a significant
(P < 0.05) increase in serum phosphate levels when
patients with undetectable 1,25-(OH)2D levels were compared
to patients with normal and low 1,25-(OH)2D levels, but
phosphate levels were still within the normal range (Fig. 1B). Serum
levels of 1,25-(OH)2D and phosphate in HIV-infected
patients were inversely correlated (r = -0.49; P
< 0.001).
Calcitonin levels were within the normal range (Table 1), and there
were no significant differences in serum calcitonin between
HIV-infected patients with undetectable 1,25-(OH)2D serum
levels and other patients (data not shown)
Clinical manifestations
As shown in Fig. 2, symptomatic
HIV-infected patients (CDC groups B and C) had significantly lower
serum levels of 1,25-(OH)2D compared to controls
(P < 0.05), with the lowest values in CDC group C. In
CDC group C, eight severely immunodeficient patients (CD4+
lymphocyte count range, 1–28 x 106 cells/L) had
chronic, ongoing events; 6 of these patients (2 MAC, 3 CMV, and 1
hepatitis C) had undetectable 1,25-(OH)2D levels, and 1
(MAC) had a serum level below the normal range.
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When patients with undetectable 1,25-(OH)2D levels
were compared to other patients, CD4+ lymphocyte counts
were significantly (P < 0.01) lower in the former
group (Fig. 1F). However, there was no strong correlation between
1,25-(OH)2D and CD4+ lymphocyte counts in the
entire patient population (r = 0.26; P =
0.06).
CD8+ lymphocyte counts were not significantly different in
HIV-infected patients and controls (Table 1) and did not change
significantly with different 1,25-(OH)2D levels (Fig. 1G).
Sustained TNF activation is a distinctive feature of the persistent
immune activation seen in HIV infection (13), as also demonstrated in
the present study (Table 1). Serum levels of TNF were significantly
higher in patients with undetectable 1,25-(OH)2D (Fig. 1H),
and there was a strong negative correlation (r = -0.55;
P < 0.001) between serum levels of TNF and
1,25-(OH)2D in HIV-infected patients.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The rate of 1-hydroxylation of 25D in the kidney normally determines
the serum level of 1,25-(OH)2D, and the hydroxylation step
is tightly regulated and influenced by a number of factors. Therefore,
a reasonable assumption would be that inadequate 1-hydroxylation
might be a cause of 1,25-(OH)2D deficiency in HIV-infected
patients. Normally, PTH as well as calcitonin stimulate
1,25-(OH)2D production, and low serum levels of these
substances would decrease the rate of 1-hydroxylation (3, 16) (Fig. 3). However, in HIV-infected patients,
calcitonin levels are within the normal range, and although there was a
slight reduction in PTH, serum levels of these parameters are not lower
in patients with undetectable 1,25-(OH)2D than in other
patients. This indicates that there may be some defect in PTH
production/secretion in HIV-infected patients, but altered PTH
production can not in itself explain the low 1,25-(OH)2D
serum levels. HIV-infected patients with undetectable
1,25-(OH)2D concentrations had higher phosphate levels than
the other patients, and high phosphate levels may inhibit
1-hydroxylation of 25D. Yet, even if serum phosphate was
significantly increased in patients with undetectable
1,25-(OH)2D levels, it was still within the normal range,
so this cannot be the whole explanation for the decreased
1,25-(OH)2D levels. Although HIV-infected patients in the
present study had serum creatinine levels within normal limits, a
subclinical renal dysfunction affecting hydroxylation cannot be
excluded.
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-hydroxylase activity is in some way inhibited in HIV-infected
patients. Low 1,25-(OH)2D levels would normally stimulate
1-hydroxylase activity (3), and the lack of this feedback regulation
further suggests an impairment of 1-hydroxylase activity. The reason
for this is unclear, but several not mutually exclusive factors may be
involved. In vitro studies have demonstrated an effect of
TNF on 1-hydroxylation in both renal and other tissues (17, 18, 19).
Bikle et al. found that TNF could both inhibit and
enhance hydroxylase activity in keratinocytes, at least partly
dependent on the degree of differentiation of these cells (18).
Furthermore, TNF has been found to induce vitamin D 1-hydroxylase
activity in human macrophages (20, 21) and may be involved in the
pathogenesis of hypercalcemia sometimes seen in human sarcoidosis and
tuberculosis. However, during HIV infection there is a down-regulation
of TNF receptors on monocytes/macrophages in advanced clinical disease
(22), and granuloma formation is commonly not seen in mycobacterial
infections in these patients. Thus, we suggest that this up-regulation
of vitamin D 1-hydroxylase activity may not necessarily be operative in
HIV-infected individuals. Of particular interest, TNF appears to
impair the stimulatory effect of PTH through mechanisms involving
down-regulation of PTH receptors, impairment of protein kinase C
activity, and inhibition of cAMP response after PTH stimulation (19, 21). Thus, although the exact role of enhanced TNF activity in the
induction of 1,25-(OH)2D deficiency remains to be
clarified, it may well contribute to 1,25-(OH)2D deficiency
in these patients at least partly by blocking the PTH stimulatory
effect on vitamin D 1-hydroxylase. The strong correlation between
elevated TNF levels and decreased 1,25-(OH)2D levels in
HIV-infected patients is therefore interesting, and it is conceivable
that this correlation may reflect an inhibitory effect on hydroxylation
of 25D, possibly through blocking of the PTH effect. In addition to a possible impairment of 1,25-(OH)2D production, another explanation for low serum levels of 1,25-(OH)2D may be increased degradation of 1,25-(OH)2D. 1,25-(OH)2D is important for cellular differentiation, especially for cells in the immune system (10, 23). HIV infection is characterized by an extremely high turnover of T lymphocytes (24). It is not inconceivable that increased need for 1,25-(OH)2D for maturation of lymphocytes may account for at least part of the decreased serum levels of 1,25-(OH)2D, or that there is a faster turnover of 1,25-(OH)2D in immunoactivated, rapidly proliferating T cells.
Under normal conditions 1,25-(OH)2D and PTH produce their net effects on calcium metabolism through complex interactions (3, 25), and 1,25-(OH)2D deficiency is usually associated with secondary hyperparathyroidism, hypocalcemia, and subsequent decalcification of bone (4, 5, 26). Low levels of 1,25-(OH)2D are traditionally believed to increase PTH production (27), and in vitro studies have shown that 1,25-(OH)2D suppresses PTH production by directly suppressing PTH gene transcription (28). This pattern was not seen in HIV-infected patients, in whom the effect of 1,25-(OH)2D deficiency on serum calcium levels was almost negligible. Furthermore, instead of hyperparathyroidism, these patients had slightly decreased PTH levels, in accordance with some other reports studying PTH metabolism in HIV infection (29, 30). However, recent studies have established that a calcium ion-sensing cell surface receptor may be the main regulator of PTH production (3, 31), and it is therefore possible that low 1,25-(OH)2D levels do not stimulate PTH production as long as the serum calcium level remains normal.
The combination of markedly depressed 1,25-(OH)2D levels
and normal or only slightly decreased serum calcium may seem
surprising. However, the independent role of 1,25-(OH)2D in
the regulation of serum calcium has recently been questioned (3, 32),
and it is possible that the effect of 1,25-(OH)2D
deficiency on serum calcium levels is not exhibited until stressed by
reduced calcium availability. Nevertheless, normal or only slightly
decreased serum calcium in HIV-infected patients with persistently
reduced 1,25-(OH)2D levels indicate that compensatory
mechanisms make calcium available for the extracellular compartment.
Increased mobilization of calcium from bone may be one such mechanism,
and indeed, increased bone resorption has been suggested in
HIV-infected patients and other retroviral infections (33, 34). Again,
this may be related to increased levels of TNF and other
proinflammatory cytokines (35).
In addition to its role in calcium homeostasis, 1,25-(OH)2D influences the immune system both in vivo and in vitro. A substantial amount of experimental data from in vitro studies have established 1,25-(OH)2D as a potent modulator of cells of the immune system (2, 10, 11, 36). In vivo, vitamin D deficiency has been associated with macrophage dysfunction and bacterial infections (8, 9, 36, 37, 38, 39). Furthermore, until the advent of antibiotics, vitamin D was used to treat mycobacterial infections (6, 7), and infants with hereditary vitamin D-dependent rickets may die of serious pulmonary infections if the condition is not recognized and treated (5). Also, administration of 1,25-(OH)2D to uremic patients has been shown to improve immune functions and modulate cytokine secretion (4, 40). Thus, even if the effect of severe 1,25-(OH)2D deficiency on serum calcium is negligible in these patients, the correlation between low 1,25-(OH)2D serum levels and clinically advanced HIV infection supports other in vivo observations that low 1,25-(OH)2D serum levels may contribute to immunodeficiency.
Our observation of normocalcemic 1,25-(OH)2D deficiency is
not unique. Careful review of the literature reveals conditions with
similar metabolic patterns (41, 42, 43, 44) (Table 2). It is interesting that
1,25-(OH)2D deficiency with only minor alterations in serum
PTH and calcium levels may be present in a number of conditions with
marked immunological hyperactivity, such as tuberculosis, septicemia,
and myelomatosis, as well as in AIDS. This is in sharp contrast to the
pattern seen in severe renal failure (Table 2). In all reports
concerning conditions with immunological hyperactivity and
1,25-(OH)2D deficiency, there seems to be an unknown factor
contributing to inhibition of the 1-hydroxylase activity. It is
tempting to speculate that this factor may be related to activity of
TNF or other proinflammatory cytokines, which are known to be
increased in septicemia, HIV infection, and certain malignancies.
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and 1,25-(OH)2D have been observed at the cellular level
(45, 46, 47), and enhanced TNF levels may impair the action of
1,25-(OH)2D on various leukocytes. The low
1,25-(OH)2D and high TNF levels observed here may
therefore further impair the immune response in HIV-infected patients
both independently and in combination and may represent an important
feature of the pathogenesis of HIV-related immunodeficiency. The
combination of low 1,25-(OH)2D and high TNF levels may
also contribute to the impairment of immune function in other diseases
characterized by markedly depressed 1,25-(OH)2D serum
levels and immunological hyperactivity. Thus, the findings in the
present study may not only represent a previously unrecognized
mechanism for induction of 1,25-(OH)2D deficiency in human
disease, but may also reflect the importance of adequate serum levels
of 1,25-(OH)2D for satisfactory performance of the immune
system in man.
Received April 10, 1998.
Revised July 9, 1998.
Accepted August 6, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxylase regulation: induction by inflammatory
cytokines and suppression by dexamethasone and uremia toxin. J
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regulation of 1,25-dihydroxyvitamin D
production by human keratinocytes. Endocrinology. 129:33–38.[Abstract]
induces vitamin D-1-hydroxylase activity
in normal human alveolar macrophages. J Cell Physiol. 142:652–656.[CrossRef][Medline]
modulates parathyroid hormone action in UMR-106–01
osteoblastic cells. J Bone Miner Res. 8:1191–1200.[Medline]
(TNF ) and
TNF receptors (TNFRs) on mononuclear cells in human immunodeficiency
virus type 1 infection: low percentage of p75-TNFR positive cells in
patients with advanced disease and high viral load. Blood. 90:2670–2679.,25-dihydroxyvitamin D. N Engl
J Med. 289:817–822.
-hydroxyvitamin D3 on cell-mediated immunity
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: structural determinants
within the vitamin D response element. Endocrinology. 134:2524–2531.[Abstract]
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