Regulation of 1,25-dihydroxyvitamin D3 production by cultured alveolar macrophages from normal human donors and from patients with pulmonary sarcoidosis

Regulation of the production of the biologically active vitamin D3 sterol 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by cultured pulmonary alveolar macrophages (PAM) obtained from 6 patients with pulmonary sarcoidosis and from 9 normal subjects was studied. The sarcoid cells, all collected from patients with normal calcium metabolism, synthesized 1,25-(OH)2-[3H]D3 from the substrate 25-hydroxyvitamin [3H]D3 (25OH-[3H]D3), whereas in vitro incubation with recombinant human interferon-gamma (IFN gamma) or lipopolysaccharide (LPS) was required for induction of synthesis of the hormone by normal PAM. Exogenous 1,25-(OH)2D3 (10-100 nmol/L) decreased endogenous hormone production by normal PAM by approximately 45%. The relative inhibitory effect of 1,25-(OH)2D3 was less pronounced in sarcoid PAM, in which 10-100 nmol/L 1,25-(OH)2D3 inhibited 250HD3-1-hydroxylase by approximately 25%. An accompanying induction of the 250HD3-24-hydroxylase, which is typical for renal cells, was found at low levels in only 3 of 10 experiments; in this regard, no differences between sarcoid and normal PAM were apparent. PTH or forskolin did not influence 250HD3 metabolism by PAM. 1,25-(OH)2D3 production by sarcoid PAM was enhanced by lipopolysaccharide and IFN gamma. Likewise, recombinant human interleukin-2 stimulated 1,25-(OH)2D3 production by sarcoid PAM, suggesting a possible role for both IFN gamma and interleukin-2 in the induction of 1,25-(OH)2D3 synthesis by sarcoid PAM in vivo. Recombinant human IFN alpha, IFN beta, and granulocyte-macrophage colony-stimulating factor had little effect. Dexamethasone and chloroquine, which have in vivo antihypercalcemic activity in sarcoidosis, both inhibited 1,25-(OH)2D3 synthesis by sarcoid PAM; chloroquine simultaneously stimulated the 24-hydroxylase. Our studies suggest that the 250HD3-metabolizing system in PAM is in some respects different from renal metabolism of 250HD3.     

A role for endogenous arachidonate metabolites in the regulated expression of the 25-hydroxyvitamin D-1-hydroxylation reaction in cultured alveolar macrophages from patients with sarcoidosis

In the human granulomatous disease sarcoidosis hypercalcemia and/or hypercalciuria result from the endogenous overproduction of 1,25-dihydroxyvitamin D [1,25-(OH)2D] by the disease-activated macrophage. Unlike the renal 25-hydroxy-vitamin D (25OHD)-1-hydroxylase, normally the sole synthetic source of the hormone in man, the 25OHD3-1-hydroxylation reaction in cultured pulmonary alveolar macrophages (PAM) from patients with sarcoidosis is subject to stimulation by the immune cytokine interferon-gamma (IFN gamma) and inhibition by the antiinflammatory glucocorticoid dexamethasone. The data presented here suggest that IFN gamma and calcium ionophore A23187 promote enhanced expression of the sarcoid PAM 25OHD3-1-hydroxylation reaction by increasing endogenous arachidonic acid metabolism through the 5-lipoxygenase pathway. Dexamethasone, an inhibitor of the cellular phospholipase-A2-arachidonic acid-generating system, and BW755C, a lipoxygenase pathway inhibitor, inhibited PAM 1,25-(OH)2D3 synthesis by 64% and 54%, respectively. Conversely, leukotriene C4, a distal metabolite in the arachidonic acid 5-lipoxygenase pathway, increased the hydroxylation reaction by 234% and restored dexamethasone-inhibited PAM 1,25-(OH)2D3 synthetic activity. The results of this study provide presumptive evidence for an important role of agonist (IFN gamma)-calcium-modulated eicosanoid metabolism in the regulated synthesis of 1,25-(OH)2D by PAM in sarcoidosis.     

Ketoconazole decreases the serum 1,25-dihydroxyvitamin D and calcium concentration in sarcoidosis-associated hypercalcemia

Ketoconazole is an antifungal agent capable of inhibiting human steroid hormone synthesis, including renal 1,25-dihydroxyvitamin D [1,25-(OH)2D] synthesis. The ability of this drug to inhibit the extrarenal production of 1,25-(OH)2D, as occurs in human granuloma-forming disease states, including sarcoidosis, has not been evaluated. We examined the effect of ketoconazole on the 1,25-(OH)2D-calcium homeostatic mechanism in a hypercalcemic patient with sarcoidosis and on the synthesis of 1,25-(OH)2D3 in vitro by cultured pulmonary alveolar macrophages (PAM) from this and another host. Oral ketoconazole therapy (800 mg/day) decreased the serum 1,25-(OH)2D concentration 73% within 4 days; this was associated with a 15% decrease in the serum calcium concentration and a 57% decrease in the fractional urinary calcium excretion rate. In vitro, ketoconazole had a rapid onset, concentration-dependent inhibitory effect on sarcoid PAM 1,25-(OH)2D3 synthesis (ED50 = 0.1 mumol/L) that was not reversible by exposure to leukotriene C4, a potent stimulator of PAM 1,25-(OH)2D3 synthesis. Kinetic analysis of ketoconazole's action on the macrophage 1 alpha-hydroxylation reaction was examined at concentrations achieved in vivo when the drug is given orally. The velocity of the 1 alpha-hydroxylation reaction at ketoconazole concentrations of 0.01-1.0 mumol/L increased as the concentration of substrate 25-hydroxyvitamin D3 increased from 12-2000 nmol/L.     

High level of interferon gamma in tuberculous pleural effusion

It has been observed that T-lymphocytes of patients with tuberculosis produce interferon gamma (IFN gamma) in vitro. Based on this idea, we studied IFN gamma in pleural fluid and serum. We studied 80 patients with pleural effusion; 30 patients with tuberculous pleurisy had high IFN gamma concentrations in pleural fluid. Patients with malignant pleural effusions, nonspecific pleural effusion, parapneumonic effusions and pleural transudates had low levels. The IFN gamma levels were higher in those with massive tuberculous effusion and apparent pulmonary lesion on x-ray film. We found that the T4/T8 lymphocyte ratio was higher in pleural fluid than in peripheral blood. Numbers of T3 and T4 lymphocytes were higher in tuberculous pleural effusions compared with those in other patients. There is no correlation between IFN gamma levels and lymphocyte subsets in pleural effusion. Perhaps pleural T-lymphocytes produce IFN gamma after stimulation by mycobacterial antigens and this lymphokine activates macrophages, increasing their bactericidal activity against Mycobacterium tuberculosis.     

Isolation and structural identification of 1,25-dihydroxyvitamin D3 produced by cultured alveolar macrophages in sarcoidosis

Hypercalcemia and hypercalciuria in sarcoidosis are thought to result from the endogenous overproduction of an active vitamin D metabolite. We employed primary cultures of pulmonary alveolar macrophages from two patients with biopsy-proven pulmonary sarcoidosis and a recent or current clinical abnormality in calcium metabolism to synthesize in vitro a 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-like metabolite from 25-hydroxyvitamin D3 (25OHD3). The macrophage metabolite cochromatographed with [3H]1,25-(OH)2D3 on normal phase and reverse phase high performance liquid chromatography and was bound with high affinity by the chick intestinal receptor for 1,25-(OH)2D3. On UV spectroscopy, the metabolite possessed the carbon-5,7,10 (19) cis-triene chromophore characteristic of a vitamin D sterol. Electron impact mass spectrometry of trimethylsilyl ether derivatives of the metabolite revealed a mass fragmentation pattern similar to that of the trimethylsilyl ether derivative of authentic 1,25-(OH)2D3. The incubation of cultured macrophages from two patients with idiopathic pulmonary fibrosis and two with scleroderma with [3H]25OHD3 did not result in production of a metabolite with the chromatographic identity of 1,25-(OH)2D3. These data indicate that the metabolite of 25OHD3 synthesized by sarcoid macrophages in vitro is 1,25-(OH)2D3 and that the macrophage is a synthetic source of the sterol metabolite in sarcoidosis.     

Expression of 1,25(OH)2D3 receptors on alveolar lymphocytes from patients with pulmonary granulomatous diseases.

1,25(OH)2D3 is known to be produced at sites of granulomatous reactions. In order to characterize the cell types that are targets for this immunoregulatory hormone, we have evaluated the expression of 1,25(OH)2D3 receptors on peripheral blood T-lymphocytes and those recovered from the lung by bronchoalveolar lavage from patients with pulmonary granulomatous diseases (tuberculosis and sarcoidosis) and from normal control subjects using combined autoradiographic and immunohistochemical techniques. Lavage T-lymphocytes from patients with tuberculosis or with sarcoidosis, but not those from normal control subjects, expressed 1,25(OH)2D3 receptors as demonstrated by binding of [3H]1,25(OH)2D3, which was inhibited by the presence of excess unlabeled 1,25(OH)2D3, but not by the presence of unlabeled 25(OH)D3 (receptor-positive lymphocytes: sarcoidosis, 20 +/- 12%; tuberculosis, 31 +/- 17%). In contrast, blood lymphocytes from patients with granulomatous diseases did not express detectable 1,25(OH)2D3 receptors. The percentage of lavage T-lymphocytes expressing 1,25(OH)2D3 receptors was significantly greater for patients with tuberculosis presenting with isolated hilar adenopathy than for patients with pulmonary infiltrates and/or cavities. 1,25(OH)2D3 receptors were expressed to a greater extent on CD8+ T-lymphocytes than on CD4+ T-lymphocytes in sarcoidosis, whereas a greater proportion of CD4+ than of CD8+ T-lymphocytes from patients with tuberculosis were receptor-positive. These findings support the conclusion that the interaction of 1,25(OH)2D3 with its receptor on T-lymphocytes may play an important role in the regulation of granulomatous reactions, but because these receptors are expressed on different lymphocyte populations, the net effect of this potent immunoregulatory molecule is likely different in sarcoidosis and tuberculosis.     

Lymphocyte proliferation and gamma-interferon production after "in vitro" stimulation with PPD. Differences between tuberculous and nontuberculous pleurisy in patients with positive tuberculin skin test

T-lymphocytes previously sensitized by an antigen undergo blastic transformation and produce IFN tau when stimulated by the same antigen. We studied the lymphoblastic response to PPD and IFN tau production in pleural fluid and peripheral blood of 41 patients (15 with tuberculous pleural effusion, 13 with nontuberculous pleurisy and positive tuberculin skin test, and 13 with tuberculin-negative nontuberculous pleurisy). In tuberculous pleuritis, pleural lymphocyte blastic response and IFN tau production were higher than those of peripheral lymphocytes, whereas in tuberculin-positive nontuberculous patients, peripheral lymphocyte response and IFN tau production were higher than those of pleural lymphocytes. Tuberculous pleural fluid lymphocytes underwent greater blastic transformation and produced more IFN tau than pleural lymphocytes of tuberculin-positive nontuberculous patients, whereas the opposite occurred in peripheral lymphocytes. In tuberculin-negative nontuberculous patients, there was no lymphoblastic response in either the pleural fluid or peripheral blood. These results concur with the concept of immunologic compartmentalization. In tuberculous pleuritis, there would be clonal expansion of PPD-responding T-lymphocytes in the pleural compartment. This expansion of PPD-specific lymphocytes would not occur in nontuberculous pleuritis, but lymphocytes sensitized to other antigens would accumulate in the pleural compartment.     

Enhanced production rate of 1,25-dihydroxyvitamin D in sarcoidosis

We determined the metabolic clearance and production rates of 1,25-dihydroxyvitamin D [1,25-(OH)2D] in 5 patients with sarcoidosis who had either hypercalciuria or hypercalcemia to examine whether abnormalities in the metabolism of this hormone existed. The mean MCR in the 5 patients with sarcoidosis [40 +/- 9 (+/- SD) mL/min] was similar to that in 13 normal subjects (37 +/- 6 mL/min) and that in 9 patients with absorptive hypercalciuria and renal stones (35 +/- 4 mL/min). However, the mean serum 1,25-(OH)2D concentration was significantly higher in the patients with sarcoidosis (211 +/- 60 pmol/L) than in either of the other 2 groups. The mean 1,25-(OH)2D production rate was markedly elevated in the patients with sarcoidosis (12.4 +/- 5.3 mumol/day), being more than 2-fold greater than the normal mean value (5.4 +/- 1.2 mumol/day). The highest production rates were found in patients with hypercalcemia, whereas subjects with hypercalciuria had production rates comparable to those in the patients with absorptive hypercalciuria. These data indicate that there is no impairment in the clearance of 1,25-(OH)2D in patients with sarcoidosis and that the elevated serum 1,25-(OH)2D levels are due to an increase in its production rate.     

Vitamin D metabolite-mediated hypercalcemia

The endogenous overproduction of active vitamin D sterols plays a central causative role in the hypercalcemic/hypercalciuric state associated with granuloma-forming diseases, most notably sarcoidosis, as well as with some human lymphomas. In sarcoidosis, the offending metabolite is most likely 1,25-(OH)2-D and the synthetic source is the disease-activated macrophage. About 50% of hypercalcemic patients with lymphoma harbor frankly elevated or inappropriately high serum 1,25-(OH)2-D concentrations. The source of the hormone in patients with lymphoma is not yet known. The endogenous synthesis of 1,25-(OH)2-D in patients with active sarcoidosis and lymphoma is not subject to regulation by those factors that normally control the production of 1,25-(OH)2-D by the renal 25-OH-D-1-hydroxylase. Treatment and prevention of vitamin D metabolite-mediated hypercalcemia/hypercalciuria consist of pharmacologic inhibition of the abnormal 1-hydroxylation reaction and limitation of substrates for the reaction. The former is best accomplished by the administration of anti-inflammatory concentrations of glucocorticoids and the latter by controlling vitamin D intake and sunlight exposure in susceptible hosts.     

Update on tuberculous pleural effusion

The possibility of tuberculous pleuritis should be considered in every patient with an undiagnosed pleural effusion, for if this diagnosis is not made the patient will recover only to have a high likelihood of subsequently developing pulmonary or extrapulmonary tuberculosis Between 3% and 25% of patients with tuberculosis will have tuberculous pleuritis. The incidence of pleural tuberculosis is higher in patients who are HIV positive. Tuberculous pleuritis usually presents as an acute illness with fever, cough and pleuritic chest pain. The pleural fluid is an exudate that usually has predominantly lymphocytes. Pleural fluid cultures are positive for Mycobacterium tuberculosis in less than 40% and smears are virtually always negative. The easiest way to establish the diagnosis of tuberculous pleuritis in a patient with a lymphocytic pleural effusion is to generally demonstrate a pleural fluid adenosine deaminase level above 40 U/L. Lymphocytic exudates not due to tuberculosis almost always have adenosine deaminase levels below 40 U/L. Elevated pleural fluid levels of γ‐interferon also are virtually diagnostic of tuberculous pleuritis in patients with lymphocytic exudates. In questionable cases the diagnosis can be established by demonstrating granulomas or organisms on tissue specimens obtained via needle biopsy of the pleura or thoracoscopy. The chemotherapy for tuberculous pleuritis is the same as that for pulmonary tuberculosis.     

1,25-Dihydroxyvitamin D3 antagonizes interferon-gamma-induced expression of class II major histocompatibility antigens on thyroid follicular and testicular Leydig cells

Interferon-gamma (IFN gamma) induces production and expression of major histocompatibility complex class II molecules on both marrow-derived and nonbone marrow-derived cell types. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], a seco-steroid derived from vitamin D3, has previously been reported to enhance such expression alone or together with IFN gamma on a number of monocyte/macrophage tumorigenic lines. In contrast, the present studies have found that 1,25-(OH)2D3 inhibited the ability of IFN gamma to induce class II antigen expression on nontransformed rat thyroid follicular epithelial cells (FRTL-5) and mouse testicular Leydig cells (TM3). Although 1,25-(OH)2D3 inhibited the induction of both IA and IE class II locus products, IFN gamma augmentation of class I major histocompatibility complex antigens was not affected. 1,24-(OH)2D3 and 24,25-(OH)2D3 also inhibited class II induction by IFN gamma. Notably, the relative inhibitory ability of these compounds paralleled the strength of their binding affinities for the 1,25-(OH)2D3 receptor, indicating that this antagonistic effect probably requires receptor-ligand interaction. Other steroid hormones, such as hydrocortisone or testosterone, had no inhibitory effect on IFN gamma-induced class II expression on Leydig cells. Additionally, the failure of indomethacin to reverse the effect of 1,25-(OH)2D3 and the finding that exogenous prostaglandin E2 did not inhibit class II induction in these cells indicated that prostaglandins are probably not responsible for this anti-IFN gamma activity. In total, these results suggest that an endocrinological mediator is capable of inhibiting class II induction on resident endocrine tissue populations and, therefore, could help to diminish local CD4+ T-cell recognition of these cells.     

Inhibition by prostaglandin E1 and E2 of 1,25-dihydroxyvitamin D3 synthesis by synovial fluid macrophages from arthritic joints.

Previous work has shown that renal metabolism of 25-dihydroxyvitamin D3 (25(OH)D3) to the active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is stimulated by prostaglandin E2 and inhibited by acetylsalicylate (aspirin). As prostaglandins are primary inflammatory mediators and synovial fluid macrophages are known to synthesise 1,25(OH)2D3 in vitro, the effects of prostaglandin E1, prostaglandin E2, and aspirin on the metabolism of 25(OH)D3 by cells cultured from synovial fluid of patients with inflammatory arthritis were investigated. Most cultures contained non-proliferating macrophages which formed 1,25(OH)2D3; however, two of 13 cultures contained colonies of rapidly proliferating fibroblast-like cells which formed 24,25(OH)2D3 (24,25(OH)2D3). Prostaglandin E1 and prostaglandin E2 (0.01-10 mumol/l) induced marked inhibition of 1,25(OH)2D3 synthesis (up to 94%) in a dose dependent manner after preincubations of 24 hours but not over straightforward six hour incubations. Exposure of macrophages to aspirin (1 mumol/l-1 mmol/l) for 24 hours did not affect 1,25(OH)2D3 synthesis unless the cells had been pretreated with lipopolysaccharides, in which instance 1 mM aspirin increased 1,25(OH)2D3 synthesis. Lipopolysaccharide is a macrophage activating factor which stimulates macrophages to form 1,25(OH)2D3, and it also induces prostaglandin synthesis which would be inhibited by aspirin. Taken together these results suggest that prostaglandin E1 and prostaglandin E2 synthesised by macrophages may act in an autocrine manner to attenuate the ability of macrophage activating factors, such as lipopolysaccharide, to stimulate 1,25(OH)2D3 synthesis. Prostaglandins synthesised by other inflammatory cells may also inhibit 1,25(OH)2D3 synthesis in a paracrine manner. In contrast, prostaglandin E2 and aspirin had limited effects on fibroblast 24,25(OH)2D3 synthesis. This study shows that the effects of prostaglandin E1, prostaglandin E2, and aspirin in macrophages contrast with those previously reported for the renal 25(OH)D3-1alpha-hydroxylase, where prostaglandin E2 stimulated and aspirin inhibited enzyme activity. These results further emphasise that synthesis of 1,25(OH)2D3 in non-renal sites is independently regulated, which is consistent with it having an immunological role at a local level rather than playing a part in systemic calcium homeostasis.     

Differential metabolism of 25-hydroxyvitamin D3 by cultured synovial fluid macrophages and fibroblast-like cells from patients with arthritis.

Differential metabolism of 25-hydroxyvitamin D3 (25(OH)D3) has been shown for macrophages and fibroblast-like cells (possibly synoviocytes) cultured for two to 50 days after isolation from the synovial fluid of 12 patients with various forms of inflammatory arthritis. Macrophages synthesised the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the synthesis of which was increased by bacterial lipopolysaccharide, a known macrophage activating factor. In contrast, fibroblast-like cells formed 24, 25-dihydroxyvitamin D3 (24,25(OH)2D3), synthesis of which was stimulated by 1,25(OH)2D3 and inhibited by lipopolysaccharide. The synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 by macrophages and fibroblast-like cells respectively was inhibited by ketoconazole, indicating that both hydroxylases are dependent on cytochrome P-450. Mean (SEM) synovial fluid and serum 25(OH)D3 concentrations were 16.7 (1.7) and 22.2 (2.6) ng/ml and those of 1,25(OH)2D3 were 29.4 (4.8) and 43.3 (4.0) pg/ml respectively. In most cases concentrations were lower in synovial fluid than in paired serum samples, but in two patients 1,25(OH)2D3 concentrations were greater in synovial fluid than in serum, suggesting local synthesis within the affected joints.     

Gamma delta T lymphocytes in human tuberculosis.

The manifestations of tuberculous infection reflect the immune response to infection. Most healthy tuberculin reactors develop protective immunity; tuberculous pleuritis reflects a resistant response manifest by mild disease, whereas advanced pulmonary and miliary tuberculosis reflect ineffective immunity. The role of gamma delta T cells was assessed in tuberculous infection by evaluating expansion of these cells from blood mononuclear cells after stimulation with Mycobacterium tuberculosis. After culture in vitro, the percentages of gamma delta+ cells were significantly greater in patients with protective and resistant immunity (tuberculin reactors, 25% +/- 4%; tuberculous pleuritis, 30% +/- 7%) than in those with ineffective immunity (advanced pulmonary tuberculosis, 9% +/- 3%; miliary tuberculosis, 2% +/- 1%). In leprosy, expansion of gamma delta+ cells was greater in immunologically resistant tuberculoid patients (32% +/- 4%) than in Mycobacterium leprae-unresponsive lepromatous patients (9% +/- 2%). M. tuberculosis-reactive gamma delta T cell lines produced interferon-gamma, granulocyte-macrophage colony-stimulating factor, interleukin-3, and tumor necrosis factor-alpha, cytokines that activate macrophages and may contribute to mycobacterial elimination. These findings suggest that gamma delta T cells contribute to immune resistance against M. tuberculosis.     

25-Hydroxyvitamin D3 metabolism by lipopolysaccharide-stimulated normal human macrophages

Cultured normal human pulmonary alveolar macrophages and peripheral blood monocyte-derived macrophages were studied for their capacity to metabolize [3H]25-hydroxyvitamin D3 (25OHD3). Incubation of macrophages with bacterial lipopolysaccharide (LPS) resulted in the conversion of [3H]25OHD3 to a more polar vitamin D3 metabolite (up to 15 pmol/10(6) cells). Untreated macrophages did not synthesize this metabolite. Several findings suggested that the metabolite was the biologically active form of vitamin D3, namely 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. (1) The metabolite comigrated with chemically synthesized 1,25-(OH)2D3 on four different high performance liquid chromatographic systems. (2) The metabolite had the same affinity for the chick intestinal 1,25-(OH)2D3 receptor as authentic 1,25-(OH)2D3. (3) The biological activity of the macrophage metabolite in vivo (stimulation of intestinal calcium absorption and bone calcium mobilization in rachitic chicks) was identical to the activity of chemically synthesized 1,25-(OH)2D3. The LPS-stimulated synthesis of the 1,25-(OH)2D3-like compound by macrophages was dose dependent in a linear fashion; a half-maximal response was typically found with 100-200 ng LPS/10(6) cells. Polymyxin B abolished the effects of LPS on 25OHD3 metabolism in macrophages. Our data suggest that LPS-stimulated macrophages can modulate, on a local level, the function of 1,25-(OH)2D3-responsive cells by releasing the 1,25-(OH)2D3-like metabolite.     

Extrarenal production of calcitriol in normal and uremic humans

We have previously reported low serum levels of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and increased 1,25-(OH)2D3 production after the administration of 25-hydryoxyvitamin D (25OHD) to anephric humans. Since normal alveolar macrophages are known to synthesize 1,25-(OH)2D3 when stimulated with gamma-interferon or lipopolysaccharide, we determined whether macrophages derived from peripheral blood monocytes could be an extrarenal source of 1,25-(OH)2D3. Our results demonstrated that macrophages from normal individuals synthesize 1,25-(OH)2D3. The apparent Km for 25OHD3 was 6.6 +/- 0.5 nM and the maximum velocity was 47.4 +/- 13.7 fmol 1,25-(OH)2D3/h.microgram DNA. The activity of this enzyme was reduced 37.2 +/- 3.1% by physiological concentrations (96 pmol/L) of 1,25-(OH)2D3 in the incubation medium. Normal macrophages further hydroxylated 1,25-(OH)2D3 to more polar metabolites, and this catabolic activity was significantly enhanced by physiological concentrations of 1,25-(OH)2D3. In chronic renal failure, peripheral macrophages exhibited an enhanced 1 alpha-hydroxylase activity (8.2 +/- 0.8 vs. 4.2 +/- 0.5 fmol 1,25-(OH)2D3/microgram DNA.h in controls) and a decreased capacity to degrade 1,25-(OH)2D3. Exogenous 1,25-(OH)2D3, in physiological concentrations, reduced 1,25-(OH)2D3 synthesis to a degree (23.6 +/- 8.5%) comparable to that observed in normal cells. 1,25-(OH)2D3 production by macrophages did not correlate with the severity of hyperparathyroidism. Moreover, human PTH-(1-34) in supraphysiological concentrations (20,000 and 100,000 ng/L) did not stimulate the 1 alpha-hydroxylase activity of macrophages from either normal or uremic subjects. These results demonstrate that 1) normal peripheral macrophages metabolize 25OHD3 and 1,25-(OH)2D3; 2) macrophages in uremia display higher rates of 1,25-(OH)2D3 synthesis and lower rates of catabolism than normal macrophages; and 3) 1,25-(OH)2D3 deficiency, but not hyperparathyroidism, may play a role in the stimulation of 1,25-(OH)2D3 production by macrophages in chronic renal failure.     

Tuberculous pleural effusion and tuberculous empyema

Tuberculous pleuritis has increased worldwide, especially in developing countries, as a consequence of human immunodeficiency virus co-infection. Tuberculous pleuritis is a delayed hypersensitivity reaction against mycobacterial antigens in the pleural space. Mycobacteria are detected in less than 50% of pleural samples, but the characteristic pleural involvement, granulomas with or without caseous necrosis, is evident in 56 to 80% of cases from samples obtained by percutaneous pleural biopsy. Of several pleural fluid parameters studied, adenosine deaminase and interferon gamma (IFN-gamma) have the best diagnostic yield, while polymerase chain reaction remains a promising test. Treatment of patients with tuberculous pleuritis is discussed. Tuberculous empyema is a rare form of tuberculous pleuritis. It consists of a purulent infection of the pleural cavity with detectable bacilli in pleural fluid. Diagnosis is easily established clinically and bacteriologically. Treatment is to adequately drain the pleural space and achieve lung reexpansion, in conjunction with antituberculous chemotherapy. The efficacy of different surgical techniques is discussed.     

Diagnostic significance of interferon-gamma in tuberculous pleural effusions

STUDY OBJECTIVES: Tuberculosis (TB), the single most frequent infectious cause of death worldwide, also is a major cause of pleural effusion, which in TB usually has lymphocytic and exudative characteristics. Differential diagnosis between TB and nontuberculous pleural effusion can be sometimes difficult, representing a critically important clinical problem. METHODS: We studied 46 patients presenting with pleural effusion to the National Sanyo Hospital between April 2000 and January 2001 (34 men and 12 women; mean age, 64 years). Ten patients (22%) had tuberculous pleurisy, 19 patients (41%) had malignant pleuritis, and 17 patients (37%) had pleural effusion due to an etiology other than tuberculosis or cancer. Pleural fluid concentrations of four suggested markers were measured using commercially available kits. RESULTS: The pleural fluid levels (mean +/- SE) of adenosine deaminase (83.3 +/- 18.2 U/L vs 25.8 +/- 20.4 U/L, p < 0.0001), interferon-gamma (137 +/- 230 IU/mL vs 0.41 +/- 0.05 IU/mL, p < 0.0001), immunosuppressive acidic protein (741 +/- 213 micro g/mL vs 445 +/- 180 micro g/mL, p < 0.001) and soluble interleukin 2 receptor (7,618 +/- 3,662 U/mL vs 2,222 +/- 1,027 U/mL, p < 0.0001) were significantly higher for tuberculous pleuritis than for other causes of effusion. Receiver operating characteristic analysis demonstrated that pleural fluid content INF-gamma was the best indicator of tuberculous pleurisy among four relevant biological markers. CONCLUSIONS: INF-gamma in pleural fluid is the most sensitive and specific among four biological markers for tuberculous pleuritis. Thus, our results suggest that determination of INF-gamma at the onset of pleural effusion is informative for the diagnosis of tuberculous pleuritis. Further studies including larger numbers of patients are needed to verify this result.     

Comparison of six biological markers for the diagnosis of tuberculous pleuritis

STUDY OBJECTIVE: We sought a marker to differentiate tuberculous pleural effusions from nontuberculous pleural effusions, which otherwise can be difficult. PATIENTS: We studied 55 patients with pleural effusions, 20 (36%) with tuberculous pleuritis and 35 (64%) with a nontuberculous etiology. MEASUREMENTS AND RESULTS: Pleural fluid levels of adenosine deaminase, interferon (INF)-gamma, interleukin (IL)-12p40, IL-18, immunosuppressive acidic protein, and soluble IL-2 receptors were measured and were subjected to receiver operating characteristic analysis. INF-gamma had the greatest sensitivity and specificity for tuberculous pleuritis among the six biological markers studied. CONCLUSION: The determination of INF-gamma levels in pleural fluid is the most informative in the diagnosis of tuberculous effusion.     

Tumour necrosis factor-alpha in comparison to adenosine deaminase in tuberculous pleuritis

BACKGROUND: Adenosine deaminase (ADA) is already used for the differential diagnosis of tuberculosis pleurisy. Tumour necrosis factor-alpha (TNF) is another marker which has been investigated for this purpose. OBJECTIVE: We evaluated the diagnostic value of pleural fluid and serum TNF concentrations in tuberculous pleuritis and compared them to ADA. METHODS: Sixty-two patients (24 tuberculous pleuritis, 38 non-tuberculous pleuritis) with exudative pleurisy were included. Serum and pleural fluid TNF concentrations were determined in all patients and ADA activity in 54 patients. Pleural fluid TNF concentrations and pleural fluid/serum TNF were compared to pleural fluid ADA activity and pleural fluid/serum ADA. RESULTS: When the tuberculous and non-tuberculous groups were compared, pleural fluid TNF concentrations (65.4 +/- 136.9 pg/ml vs. 54.5 +/- 144.2 pg/ml, respectively; p < 0.001), pleural fluid ADA activity (74.2 +/- 33.3 U/l vs. 23 +/- 16.3 U/l; p < 0.0001), pleural fluid/serum TNF (2.55 +/- 5.23 vs. 0.26 +/- 0.2; p < 0.001) and pleural fluid/serum ADA (4.58 +/- 8.14 vs. 1.15 +/- 0.7; p < 0.0001) were significantly higher in the tuberculous group. When cut-off points were assessed, 8 pg/ml and 40 U/l were found for pleural fluid TNF concentrations and pleural fluid ADA activity, respectively. Sensitivity, specificity, area under the curve were 87.5%, 76.3%, 0.772 for pleural fluid TNF concentrations and 90.9%, 89.5%, 0.952 for pleural fluid ADA activity, respectively; the difference between these areas under the curves was significant (p < 0.05). CONCLUSIONS: Pleural fluid TNF levels and pleural fluid/serum TNF were higher in tuberculous effusions than in other exudates, but their diagnostic value appears to be poorer than that of ADA.