Annals of the Rheumatic Diseases 1989; 48: 723-729

Synthesis of the active metabolite of vitamin D,1,25(OH)2D3, by synovial fluid macrophages inarthritic diseasesM E HAYES,' J DENTON,2 A J FREEMONT,2 AND E B MAWER'

From the Departments of 'Medicine and 2Rheumatology, Manchester University Medical School, Manchester

SUMMARY Synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown in cells fromknee joint synovial fluid of 20 patients with inflammatory rheumatoid disease, reactive orpsoriatic arthritis, or gout, all of which had high synovial fluid cell counts, and by cells from apatient with aseptic necrosis of a femoral condyle after short term (<24 hours) or long term(seven days) primary culture. Cells from 18 patients with inflammatory arthritis, five of whichhad low synovial fluid cell counts and cells from six patients with osteoarthritis were unable tosynthesise this metabolite from 25-hydroxyvitamin D3 (25(OH)D3). Macrophages are believed tobe the cells responsible for synthesising 1,25(OH)2D3 because these were significantly morenumerous in samples that formed 1,25(OH)2D3; they were also the predominant cell type presentin the aseptic necrosis sample and the only cell type present in preparations maintained for oneweek in monolayer culture.

Vitamin D is metabolised by sequential hydroxyla-tion steps, first to 25-hydroxyvitamin D3 (25(OH)D3)in the liver and then to either 24,25-dihydroxyvitaminD3 (24,25(OH)2D3) or the active hormonal form,1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in thekidney. The last metabolite then exerts calciumhomoeostatic actions in the intestine, bone, andrenal tubule after binding to specific intracellularreceptors.'

Peripheral blood lymphocytes and synovial tissuederived fibroblasts from patients with rheumatoidarthritis have also been shown to possess specificreceptors for 1,25(OH)2D3,2, but the role of themetabolite in these cells is not known. Furthermore,little is known of the actions of vitamin D and itsmetabolites in arthritis, though they are clearlyinvolved in normal bone and calcium metabolism,and deficiencies are associated with the develop-ment of rickets or osteomalacia.

Fairney et al have recently shown that knee jointsynovial fluid from patients with arthritic disorderscontains 25(OH)D3, 24,25(OH)2D3, and the vitaminD binding protein at about half serum concentra-

Accepted for publication 23 January 1989.Correspondence to Dr M E Hayes, Department of Medicine,University of Manchester, Stopford Building, Oxford Road,Manchester M13 9PT.

tions.4 Similarly, 1,25(OH)2D3 is also present insynovial fluid at 50-100% serum concentrations(Mawer, unpublished observations). These metabo-lites and their binding protein are probably presentbecause synovial fluid is formed as a plasma dialysatesupplemented with mucins such as hyaluronic acidand lubricating glycoproteins.

Studies of the extrarenal metabolism of 25(OH)D3have now shown that 1,25(OH)2D3 is synthesised invitro by normal human macrophages activated withinterferon gamma5 or bacterial lipopolysaccharides6;by macrophages activated by peritonitis from theperitoneal cavity of patients with renal failurereceiving continuous ambulatory peritoneal dialysis7;and by alveolar macrophages from patients withgranulomatous conditions such as sarcoidosis.8 Insarcoidosis this synthesis appears to depend on thesubstrate concentration and is not homeostaticallycontrolled, in contrast with the normal well regu-lated synthesis of 1,25(OH)2D3 in the renal tubule.Possibly, activated macrophages in one or more ofthe many forms of arthritis can also synthesise1,25(OH)2D3 within the affected joints.This study was undertaken to establish whether

cells, including macrophages, present in synovialfluid from patients with various forms of arthritis orgout are able to synthesise 1,25(OH)2D3.

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Patients and methods

Samples of synovial fluid were obtained from 45patients (details in Table 1) with knee joint effusionsattending the rheumatology outpatient clinics ofvarious local hospitals. Patients were assigned towell recognised diagnostic groups on the basis ofclinical criteria supplemented by appropriateimmunological, radiological, and crystallographicinvestigations. Six patients with symmetrical poly-arthritis or oligoarthropathies could not be assignedto a specific diagnostic group and were classifiedtogether as inflammatory arthritis not otherwisespecified.

Table 1 Details of patients studied

Number Commentsof patients

Rheumatoid arthritis 14 Three taking drugs*Reactive arthritis 7Psoriatic arthritis 5 Three taking drugstUnspecified inflammatory

arthritis 6Lymphocytic arthritis 1Gout 5Aseptic necrosis of a

femoral condyle 1Osteoarthritis 6

*One each taking gold, penicillamine, and methotrexate.tTaking methotrexate.

Table 2 Total and differential synovial fluid cell counts and(SEM)

Cells were harvested from the synovial fluidsamples by centrifugation at 2000 g for 10 minutes,resuspended in RPMI 1640 medium containing 10%fetal bovine serum, 2 mmol/l glutamine, and 50 iLg/mlstreptomycin (Flow), and cultured overnight in anatmosphere of 95% air/5% CO2 at 37°C. Cells fromnine samples were also maintained in monolayerculture for one week to select for adherent macro-phages which were positive for non-specific esterase.The non-adherent lymphocytes, polymorphs, andmacrophages were removed by exchanging theculture media 24-48 hours after plating the cells andalso when the culture media were replaced withincubation media after seven days.To assess 1,25(OH)2D3 synthesis the cells were

incubated with 100 pg of [3H]-25(OH)D3 (105 dpm,6-5 TBq/mmol, Amersham, UK) for six hours in 2ml of serum free RPMI 1640 medium supplementedwith 1-5 mg/ml of bovine serum albumin (ImmunoDiagnostika) to stabilise the vitamin D metabolitesin aqueous solution. The incubations were term-inated and extracted by addition of 5 ml ofchloroform:methanol (1:1). For each sample thechloroform extract was analysed by normal phasehigh performance liquid chromatography (HPLC)using a Zorbax-Sil column (4-6 mmx25 cm, DuPont) developed with a mobile phase of n-hexane:propan-2-ol:methanol, 110:6:4 by volume at 1-6-2ml/min. For each analysis eluent fractions werecollected and the radioactivity associated withthe substrate and 1,25(OH)2D3 was estimated by

1,25-hydroxyvitamin D? synthesis. Values are given as mean

Number Cellsll (x 10') 1,25(OH)2D3 synthesisofpatients Total Polymorphs Lymphocytes Macrophages (fglh/l0' macrophages)

1,25(OH)2D3 formerstRheumatoid arthritis 8 7-87 5-24 1-68 0-94 394 (84)

(2-01)' (2.01)' (0-33) (0-14)'..Reactive arthritis 7 12-03 8-28 1-91 153 373 (89)

(2-40)" (2.01)" (0-48) (0-24)***Gout 2a 18-8 16-17 0-38 2-26 350

b 9-22 5-90 2 12 1-20 861Non-specific inflammatory 2a 18-24 13-68 4-01 0-57 667

arthritis b 3-68 3-34 0-04 0-29 80Psoriatric arthritis 1 8-94 6-17 2-06 0-38 142Aseptic necrosis 1 2-0 0 0-05 1-95 580

Non-formerstHigh cell count 13 12-73 9-93 2-33 0-40 0

(1-44)** (1.69)* (0-54) (0-11)Low cell count 5 2-00 0-28 1-33 0-01-1-4 (range) 0

(0-16) (0-07) (0-26) 0-1 (median)Osteoarthritis 6 0-31 Predominantly synoviocytes 0

(0.13)

*p<-05, **p<O-Ol compared with low ceil count non-formers; ***p<0-01 compared with high cell count non-formers.tResults from patients were divided into 1,25(OH)2D3 formers and non-formers. The formers were subdivided into types of arthritis orgout and the non-formers into low and high cell count samples.

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Synthesis of 1,25(OH)2D3 in arthritic diseases 725

liquid scintillation counting. Standard 25(OH)I24,25(OH)2D3, and 1,25(OH)2D3 (donated by DrR Uskokovic, Hoffmann-LaRoche Inc, New JersUSA) were also chromatographed, and the ret4tion times of these markers were determinedultraviolet absorption at 265 nm. Further analysesselected chloroform extracts were carried out on Inormal phase (Zorbax-Sil) and on reverse ph;(Zorbax-ODS, 4.6 mmx25 cm) HPLC systerdeveloped with mobile phases of 2-5% methanoldichloromethane (2 ml/min) and 15% watermethanol (2 ml/min) respectively, to confirmidentity of the metabolic product. For these analy[3H]-1,25(OH)2D3 was used as a marker.

Cytocentrifuge preparations were made w40 000 cells per slide for each sample. Cells wi

50 250HD3REACTIVE 1 24,25S1H)OD3ARTHRITIS

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Fig. 1BFig. 1 Metabolism ofL?H1-25(0H)D.; in a six hour in

- vitro incubation system by synovialfluid cells harvestedfrom patients with reactive arthritis, rheumatoid arthritis, orgout (Fig. IA), and by cells (95% macrophages) harvested

_ from a patient with aseptic necrosis ofafemoral condyle1 (Fig. IB). Bars represent the amount oftritiated material in

eluentfractions obtained by high performance liquidchromatography (HPLC) analysis ofcell extracts on aZorbax-Sil column developed with n-hexane:propan-2-ol:methanol (110:6:4). Standard 25(OH)D?, 24,25(OH)2D ,and 1,25(OH)2D3 were used as markers, and detected byultraviolet absorbance at 265 nm --

identified and differential cell numbers determinedon the basis of their morphology in Giemsa stainedpreparations and their reaction for non-specificesterase with a-naphthyl propionate as substrate.9

Results

Synovial fluid samples were divided into two groupsaccording to whether or not the cells were able toform 1,25(OH)2D3. Cells from 21 patients synthe-sised [3H]-1,25(OH)2D3 after overnight culturefollowed by a six hour incubation with [3H]-25(OH)D3. The cells with this ability were found

- in eight out of 14 samples from patients with- rheumatoid arthritis, all seven of those with reactive5 arthritis, two out of five with gout, and three out of

12 with other types of arthritis (two with non-specific inflammation and one with psoriatic arthritis)

5 10HPLC RETENTION TIME (min)

Fig. lA

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726 Hayes, Denton, Freemont, Mawer

and in the patient with aseptic necrosis of a femoralcondyle (Table 2). Figure IA shows typical chro-matograms from HPLC analyses from the first threegroups; peaks of radioactivity corresponding to theprecursor [3H]-25(OH)D3 and a product cochro-matographing with 1,25(OH)2D3 are clearly shown,but there was no radioactive peak corresponding to24,25(0H)2D3. Synthesis of [3H-1,25(0H)2D3 wassimilarly shown in cells from the patient with asepticnecrosis (Fig. 1B); by all morphological and histo-chemical criteria more than 95% of these cells weremacrophages (Fig. 2). To confirm that macrophageswere responsible for 1,25(OH)2D3 synthesis cellswere placed in culture for one week before incuba-tion with [3H]-25(OH)D3 for six hours. Figure 3Ashows [3H]-1,25(0H)2D3 synthesis by macrophagesfrom a psoriatic arthritis sample maintained for oneweek in culture. The identity of the metaboliteformed by these cells was confirmed by cochromato-graphy with authentic [3HJ- 1 ,25(0H)2D3 on twofurther HPLC systems (Figs 3B and C)The non-formers included all six of the patients

with osteoarthritis, three patients with gout, six withrheumatoid arthritis, four with psoriatic arthritis,four with-unspecified arthritis, and a patient withlymphocytic arthritis.

In general terms, therefore, it appears that cellsfrom the synovial fluid of patients with non-inflam-matory arthropathies did not form 1,25(OH)2D3,whereas those fluids in which 1,25(OH)2D3 synthesisoccurred were from patients with inflammatoryarthropathies. This is clearly not a complete ex-planation of the results, however, as synovial fluidcells from 18 of the 38 patients with inflammatoryarthropathies did not synthesise 1,25(OH)2D3,whereas those from the patient with non-inflam-matory aseptic necrosis did. We have examined allthe available data on these patients to establish ifthere are any obvious variables that might distinguishformers from non-formers.

Non-formers differed from formers in one of twoways. Either they had high total cell counts like theformers but with a low proportion of macrophages(high cell count non-formers) or they had total cellcounts below 2-5xi09 cells/l (low cell count non-formers). The first group contained six patients withrheumatoid arthritis (including three taking drugs),four with psoriatic arthritis (including three takingmethotrexate), one with unspecified inflammatoryarthritis, and two with gout. The second groupconsisted of three patients with unspecified inflam-matory arthritis, one patient with lymphocytic arth-ritis, and one with gout. Interestingly, one exceptionto this generalisation was the sample from thepatient with aseptic necrosis, which was a non-inflammatory arthropathy characterised by a low

I

.._b

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_r_r

Fig. 2 Synovial fluid macrophages from the patient withaseptic necrosis ofafemoral condyle stained with Giemsa.

total cell count. This sample contained a highproportion of macrophages but few lymphocytesand no polymorphs and was able to form largeamounts of 1 ,25(OH)2D3 (Table 2). It would seem,therefore, that a major difference between formersand non-formers is the absolute number of macro-phages in the joint; however, samples from twopatients with low total cell counts had macrophagecounts of 0-84 and 1-38X 091O/ but failed to syn-thesise 1 ,25(OH)2D3.

Synthesis of 1 ,25(OH)2D3 was shown by cellsfrom high cell count samples that had been main-tained in cell culture for one week to select foradherent macrophages. Cells from six out of nine ofthese samples synthesised 1 ,25(OH)2D3 during a sixhour incubation period (50-2970 fgfh/incubation).Of the six samples that formed 1 ,25(OH)2D3 afterseven days in cell culture, three did not initially form1,25(OH)2D3; of the'se, one was from a patienttaking methotrexate and two had low macrophagenumbers. In the three samples in which 1,25(OH)2D3synthesis could not be shown the numbers ofadherent cells were low after seven days in culture.

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250HD3A

1 ,25OH)2D324,25(OH)2D3 I

11 I--I I It I III It 11I I III

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5l

5 10 151 ,25(OH)2D3C I~~~~~~2OD

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Fig. 3 Metabolism of['HI-25(OH)D? in a six hourin vitro incubation system by adherent synovialfluidmacrophagesfrom a patient with psoriatic arthritis aftermaintenance in cell culture for seven days. Bars representthe amount of tritiated material in eluent fractions obtainedby analysis ofaliquots ofthe cell extract on three differenthigh performance liquid chromatography (HPLC) systems:(A) Zorbax-Sil column developed with n-hexane:propan-2-ol: methanol (110:6:4); (B) Zorbax-Sil columndeveloped with 2 5% methanol in dichloromethane;(C) Zorbax-ODS column (reverse phase) developed with15% water in methanol. Standard 25(OH)D.,24,25(OH),D1, and 1,25(OH)2D_ detected bv ultravioletabsorbance at 265 nm (- - -) orJ?IH1-1.25(OH),D(-0-*-) were used as markers.

Synthesis of 1,25(OH),D3 in arthritic diseases 727

Discussion

These results show for the first time that synovialfluid cells are capable of synthesising the activevitamin D metabolite, 1,25(OH)2D3, when main-tained in either short term (<24 hours) or long term(seven days) primary culture. In contrast, no syn-thesis of 24,25(OH)2D3 could be shown, thoughother workers have previously reported synthesis ofthis metabolite in normal human macrophages invitro. In one such study macrophages exposed tointerferon gamma initially formed 1,25(OH)2D3,but after prolonged exposure they began to syn-thesise 24,25(OH)2D3. I Synthesis of 24,25(OH),D3has also been observed in articular cartilage and bychondrocytes cultured in vitro.' l

Although synovial fluid contains a heterogeneouspopulation of cells, with particularly high numbersof polymorphs in high cell count inflammatoryarthritis, it appears to be the macrophages thatsynthesise 1,25(OH)2D3. Macrophages were signifi-cantly more numerous in samples that formed1,25(OH)2D3 and were also the predominant celltype present in the aseptic necrosis sample and theonly cell type present in preparations maintained forone week in cell culture. Macrophages from mostforms of high cell count inflammatory arthritis andgout appeared to synthesise 1 ,25(OH)2D3. Inour study 13 cell samples did not metabolise1,25(OH)2D_l but six of these patients were receiv-ing either the antimetabolite methotrexate (one withrheumatoid arthritis, three with psoriasis), gold, orpenicillamine (both rheumatoid arthritis), whichmay have influenced cellular functions in macro-phages, including the activity of the 25(OH)D-1-hydroxylase enzyme.To form 1,25(OH)2D3 the macrophages must

presutnably be in an activated state (as in sarcoidosisand peritonitis), but it is uncertain whether thisoccurs before or after they enter the synovial fluid.In gout th.e cells are activated by uric acid crystalswithin the joint, whereas in reactive arthritis, whichdevelops secondary to systemic inflammatory condi-tions, the monocytes are probably activated beforethey enter the joint. The need for cells to beactivated may explain why two of the patients withlow cell count non-specific inflammatory arthritisdid- not form 1,25(OH)2D3 despite having highnumbers of macrophages. In the aseptic necrosissamplie the cells were probably activated by particlesof necrotic bone and thus actively syntlwsised1,25(OH)2D3.An adequate substrate concentration is another

requirement for the synthesis of J,25(OH)2D3,which is likely as 25(OH)D3 is preseft in synovialfluid at about half serum concentjratipns'4 and these

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concentrations are about 50 times greater than thosepresented to the cells in the incubation system usedin our study.The significance of the ability of macrophages

from patients with high cell count inflammatoryarthritis to synthesise 1,25(OH)2D3 is not clear. Evi-dence that the reaction may occur in vivo is providedby results showing that patients with rheumatoidarthritis respond to a single dose of 25(OH)D3(10 000 IU) by increasing serum 1,25(OH)2D3 con-centrations significantly compared with controls,'2but this rise is short lived. There is no evidence thatrheumatoid patients as a group have higher thannormal circulating concentrations of 1,25(OH)2D3as a result of extrarenal synthesis. 13 This may resultfrom feedback regulation of renal 1,25(OH)2D3synthesis' by that formed outside the kidney. Syn-thesis of 1,25(OH)2D3 mediated by macrophagesmay result in raised concentrations only when the25(OH)D3 levels are abnormally high or the mass oftissue involved is large, as in some cases ofsarcoidosis14 and malignant disease.'5Measured concentrations of 1,25(OH)2D3 in

synovial fluid are lower than in serum (Mawer,unpublished observations), probably owing to thesmaller concentration of vitamin D binding protein.The importance of 1,25(OH)2D3 production bymacrophages from arthritic patients may be greaterwhen cells are contained within the synovium andare thus adjacent to the sites of tissue damage andbone erosion within the joint.The presence of specific receptors for

1,25(OH)2D3 in normal human monocytes andactivated lymphocytes'6 17 and in peripheral bloodlymphocytes2 and synovial tissue derived fibro-blasts3 from patients with rheumatoid arthritissuggests a physiological role for the metabolife injoint disease. Furtermore, many of the reportedeffects of 1,25(0H>2D3 may be relevant to arthritis.For example this metabolite appears to inhibitproliferation of both B and T activated lymphocytesand reduces interleukin 2 production by activatedT lymphocytes in vitro.'8 1 ,25(0H)2D3 has alsobeen shown to promote differentiation of nlotiWtesinto macrophages and the fusion of macrophlaja tomultinucleated giant cells with bone rdetingactivity.20 21 This is in addition to the kno*n ailityof 1,25(OH)2D3 to inhibit collagen sn byosteoblasts and to promote bone resorption.1slast effect may be achieved by an indirect adtibn (if1,25(OH)2D3 on osteoblasts to stimulate boneresorption by osteoclasts, which unlike osteoblastsdo not appear to have receptors for 1,25(0H)2D3.23Thus within the synovial fluid and synovium1,25(OH)2D3 may have complex paracrine andimmunoregulatory functions which could influence

the development of the juxta-articular osteoporosisthat occurs in arthritis. 2 If this is the case then theuse of drugs that inhibit the extrarenal 25(OH)D-1-hydroxylase may play an important part in reducingthe extent of tissue damage and bone erosion thatoccurs in arthritis.Support for this study was provided by project grants from theMedical Research Council to Dr E B Mawer and ProfessorS Tomlinson, and by the Arthritis Research Council and NorthWest Regional Research Grants Committee to Dr A J Freemont.We are also grateful to the many clinicians who collected samples,to D Bayley and P Lockey for technical assistance, and to Dr MDavies for helpful discussions.

References

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2 Manolagas S C, Werntz D A, Tsoukas C D, Provvedini D M,Vaughan J H. 1,25-Dihydroxyvitamin DI receptors in lympho-cytes from patients with rheumatoid arthritis. J Lab Clin Med1986; 108: 596-600.

3 Sambrook P N, Eisman J A, Breit S. 1,25-Dihydroxyvitamin D3receptors in synovial cells in rheumatoid arthritis [Abstractl.'Aust NZ J Med 1987; 17: 120.

4 Fairney A, Starffen A M, May C, Seifert M H. Vitamin Dmetabolites in synovial fluid. Ann Rheum Dis 1987; 46: 370-4.

5 Koeffler H P, Reichel H, Bishop J E, Norman A W. Gamma-interferon stimulates production of 1,25-dihydroxyvitamin D3by normal human macrophages. Biochein Biophys Res Comnmun1985; 127: 596-603.

6 Reichel H, Koeffler P, Bishop J E, Norman A W. 25-Hydroxy-vitamin D3 metabolism by lipopolysaccharidc-stimulatcdnormal human macrophages. J Clin Endocrinol Metab 1987; 64:1-9.

7 Hayes M E, O'Donoghue D J 0, Ballardie F W, Mawer E B.Peritonitis induces the synthesis of la, 25-dihydroxyvitamin D3in macrophages from CAPD patients. FEBS Lett 1987; 220:307-10.

8 Adams J S, Sharma 0 P, Gacad M A. Singer F R. Metabolismof 25-hydroxyvitamin D3 by cultured pulmonary alveolarmacrophages in sarcoidosis. J Clin Invest 1983; 72: 1856-60.

9 Freemont A J, Davies J S. Acid esterase activity in lymphocytesand other cells: a comparison of six alpha-naphthyl bascdsubstrates. Med Lab Sci 1982; 39: 405-7.

10 Reichel H, Koeffler H P, Norman A W. Synthesis in vitro of1,25-hydroxyvitamin DI by interferon-gamma-stimulatcdnormal human bone marrow and alveolar macrophagcs. J BiolChem 1987; 262: 10931-7.

11 Garabedian M, Leiberherr M. N'Guycn T M, Corvol M T.Bailly Du Bois M. Balsan S. The in vitro production and activityof 24,25-dihydroxycholecalciferol in cartilage and calvarium.Clin Orthop 1978; 135: 241-8.

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14 Sandler L M, Winearls C G, Fraher L J, Clemens T L, Smith R,O'Riordan J L H. Studies of the hypercalcaemia of sarcoidosis:effect of steroids and exogenous vitamin D) on circulatingconcentrations of 1,25-dihydroxyvitamin D1. Q J Med 1984;210: 165-80.

15 Davies M, Mawer E B, Hayes M E, Lumb G A. Abnormalvitamin D metabolism in Hodgkin's lymphoma. Lancet 1985; i:1186-8.

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16 Provvedini D M, Tsoukas C D, Deftos L J, Manolagas S C.1,25-Dihydroxyvitamin D3 receptors in human leukocytes.Science 1983; 221: 1181-3.

17 Bhalla A K. Amento E P. ClemensT L, Holick M F, Krane S M.Specific high-affinity receptors for 1,25-hydroxyvitamin D. inhuman peripheral blood mononuclear cells: presence in mono-

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18 Shiozawa S, Shiozawa K, Tanaka Y, Fujita T. la,25-Di-hydroxyvitamin D3 inhibits proliferative response of T- andB-lymphocytes in serum-free culture. Int J Immunopharmacol1987; 9: 719-23.

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Synthesis of 1,25(OH)2D3 in arthritic diseases 729

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21 Bar-Shavit Z. Teitlebaum S L. Reitsma P, et al. Induction ofmonocytic differentiation and bone resorption by 1,25-di-hydroxyvitamin D3. Proc Natl Acad Sci USA 1983; 80: 5907-1 1.

22 Raisz L G, Kream B E, Smith M A, Simmons H A.Comparison of the effects of vitamin D metabolites on collagensynthesis and resorption of fetal rat bone in organ culture.Calcif Tissue Int 1980; 32: 135-8.

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