A B S T R A C T Metabolism of [3H]25-hydroxyvitamin D3(25-OH-D3) was studied in primary cultures of pulmonary alveolar macrophages (PAM) from seven patients with sarcoidosis and two patients with idiopathic pulmonary fibrosis. Production of a [3H]1,25-dihy-[3H]25-OH-D3 was detected in lipid extracts of cells from five patients with sarcoidosis. Synthesis of this compound in vitro was limited to viable PAM and was greatest in cells derived from a patient with hypercalcemia and an elevated serum concentration of 1,25-dihydroxyvitamin D. The tritiated PAM metabolite coeluted with authentic 1,25-(OH)2-D3 in three different solvent systems on straight-phase high performance liquid chromatography (HPLC) and demonstrated binding to extracted receptor for 1,25-(OH)2-D3, which was identical to that of commercially available [3H]1,25-(OH)2-D3 of comparable specific activity. Incubation of PAM with high concentrations of 25-OH-D3 resulted in production of an unlabeled metabolite that co-chromatographed with the 3H-PAM metabolite on HPLC and that was bound with high affinity by both the specific receptor for 1,25-(OH)2-D3 and antiserum to 1,25-

The hypercalcemia of sarcoidosis is thought to result from the extrarenal overproduction of an active vitamin D sterol (1, 2). We reported (3, 4) that cultured pulmonary alveolar macrophages (PAM) ~ from patients with sarcoidosis are capable of metabolizing 25-hydroxyvitamin D~ (25-OH-Ds) to la,25-dihydroxyvitamin D~ [1,25-(OH)2-Ds], suggesting that 1,25-(OH)2-D3, the naturally occurring active metabolite of vitamin D3, is a hypercalcemia-causing factor in sarcoidosis. However, the characteristics of this 25-OH-Ds-converting activity of sarcoid PAM in vitro are poorly understood. It does appear that the l ahydroxylation process is specific for cells derived from patients with sarcoidosis: 1,25-(OH)2-D~ is not synthesized by PAM from patients with other types of pulmonary disease (4). In addition, the specific activity of the cellular converting reaction is greatest in PAM derived from patients with diffuse, infiltrative pulmonary disease and clinical evidence of abnormal calcium metabolism (3, 4). The aim of the current report is to describe the kinetics of 1,25-(OH)2-D3 production by these cells in vitro and to delineate factors that may be important regulators of the conversion reaction. Abbreviations used in this paper: FCS, fetal calf serum; HPLC, high performance liquid chromatography; IFN, interferon; 1,24,25-(OH)3-D3, 1,24(R),25-trihydroxyvitamin D3; 1,25-(OH)~-D3, I a,25-dihydroxyvitamin Ds; 1 a-OH-D3, 1 a-hydroxyvitamin D3; 24,25-(OH)2-D~, 24(R), 25-dihydroxyvitamin Ds; 25-OH-Ds, 25-hydroxyvitamin Ds; 25,26-(OH)~-D~, 25,26-dihydroxyvitamin Ds; PAM, pulmonary alveolar macrophage. J. Exp. MED. Materials and Methods

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.