315 Osteoarthritis VITAMINS AND ARTHRITIS The Roles of Vitamins A, C, D, and E Interactions between all of the major joint tissues, including articular cartilage, synovium, and subchondral bone, have been implicated in the pathogenesis of osteoarthritis (OA). [The pathogenetic mechanisms underlying the development of OA are not fully understood; likewise, those factors that sustain or impede the progression of OA are poorly identified. Nevertheless, patients frequently have sought explanations for the development and progression of OA in dietary practices and nutritional deficiencies from either their health care provider or popular media. Enticing promises have been offered over the decades for inexpensive nutritional supplements that will end the individual's misery. Currently, there is much attention directed toward the use of antioxidant vitamins, and of vitamin D, as a means of preventing or ameliorating the pain and disability of OA. This attention is driven, in part, by our expanding appreciation of nutrition factors, particularly the vitamins, that contribute to oxidate processes and bone turnover that may be intrinsic to the development and progression of OA. Vitamins A, C, and, E are the major antioxidants in the diet or in dietary supplement products that have been identified as having a potential for antioxidant activity in the processes associated with OA. Vitamin D also may play an important role in OA by way of bone. 316 mineralization and cell differentiation--roles that parallel the actions of vitamin A and vitamin C. There are at least four possible pathways in which these nutrients can influence OA: protection against oxidative damage; modulation of the inflammatory response; cellular differentiation; and biologic actions related to bone and collagen synthesis. This article briefly discusses these vitamins and pathways using research derived from animal models, tissue cultures, clinical studies, and epidemiologic studies. Protection Against Oxidative Damage Oxygen radicals have been identified as potent agents in the destruction of cartilage and connective tissue. Multiple roles have been suggested. There is evidence that cells within joints produce reactive oxygen species (ROS) and that the attendant oxidative damage is physiologically important in OA. For example, ROS have been shown to cause depolymerization of hyaluronic acid, and degradation of proteoglycans and type II collagen. This seems to occur by direct action and, indirectly, by activation of latent collagenase. An increase in levels of proteolytic enzyme in the synovial fluid has been observedand is thought to be, at least in part, because of damage of the chondrocyte membrane by oxidative speciesThe roles of nutrients in the OA processes are complex because of the interactive system in which they operate. For example, reactive oxygen species are produced endogenously in normal metabolism, and exogenously as a consequence of toxins, infection, injury, radiation, and excessive exercise. Although mechanisms exist within the body to protect tissues from excessive oxidative action, production of ROS can overwhelm those modulating factors. In those instances in which anabolic and catabolic activities become unbalanced, tissue damage can result, especially in extracellular spaces where antioxidant enzymes are not as prevalent. When free radicals attack polyunsaturated fatty acids (PUFA) in membrane lipids, they create lipid peroxides, which spontaneously decompose to form lipid radicals. PUFAs, with their double bonds, are readily attacked by free radicals and oxidized to lipid peroxides, which have the capacity to damage cells. The free radicals produced by lipid peroxidation, peroxyl radicals, are capable of removing hydrogen from adjacent fatty acid side chains, thereby propagating a chain reaction known as lipid peroxidation. A single initiating event can lead to hundreds of such events. The production of free radicals also can be the consequence of tissue damage associated with arthritisOxidative stress is believed to contribute to the progression of arthritis rather than the initiation of disease. 317 Figure 1. Lipid peroxidation. ROS = reactive oxygen species; PUFA = polyunsaturated fatty acids; OA = osteoarthritis. Modulation of the Inflammatory Response Reactive oxygen species are generated by, and are an integral part of, the immune response. Antioxidants are important modulators of these responses and prevent excessive damage to tissues and cells. With injury and tissue damage, mast cells release inflammatory mediators that increase vascular permeability and permit complement and cells to enter tissues from the circulation. The mast cells also promote the activity of polymorphonuclear neutrophils (PMNs). The activities of PMNs and mast cells create an acute inflammatory response that leads to removal of damaged tissues and cells, tissue repair, and healing. Once macrophages are engaged, there is a potential for further cell-mediated response involving natural killer (NK) cells. These activities can lead to phagocytosis and chronic inflammation. Chondrocytes are normally surrounded by an extracellular matrix (ECM) and a repair process is initiated when this matrix is removed. Degradation and loss of the articular cartilage matrix is a central feature of OA. This loss of matrix is accompanied by an increased synthesis of matrix molecules, a process thought to be involved in matrix repair. In the presence of lipid peroxidation, arachidonic acid cascade products (i.e., prostaglandins, leukotrienes) release proinflammatory lipid peroxides that may cause vascular leakage, chemotaxis, and adherence 318 of polymorphonuclear leukocytes. In turn, these may promote the generation of reactive oxygen species and release of proteolytic enzymes that degrade collagen, proteoglycan, and other components of the cartilage matrix. Cellular Differentiation Vitamin A and vitamin D are now recognized as fundamental elements of cellular maturation and cellular differentiation. Retinoic acid, an oxidized form of vitamin A, regulates embryonic development and the differentiation of epithelial and mesenchymal tissues throughout life. Vitamin D has a role in the differentiation of keratocytes and monocytes into macrophages.Vitamin D also appears to act upon T-lymphocytes, which produce a variety of lymphokines, including the potent bone resorbing agent, osteoclast activating factor. Additional Biologic Actions Independent of their protective effect on ROS and modulation of the immune response, these vitamins have well-described biologic roles related to OA. For example, vitamins A and D have essential roles in bone development and maintenance of epithelial tissue, and vitamin C has an essential role in the production of collagen. Nutrients and Osteoarthritis It is noteworthy that there are multiple mechanisms through which nutrients can have an effect on either the initiation or progression of OA. For the most part, however, studies in this area have been limited to laboratory investigations in tissue culture systems and await extension into clinical or epidemiologic populations. On the other hand, the basic research data on these nutrients (and their potential for ameliorating disease) have been evoked inappropriately as scientific disguises for marketing strategies. Nonetheless, their potential importance to OA reinforces the need for well-developed investigations in both affected clinical and at-risk populations. VITAMIN A AND THE CAROTENOIDS Vitamin A occurs naturally in two forms: active, fat-soluble retinol, which is generally derived from animal tissues; and a water-soluble precursor, provitamin A, also known as beta-carotene, which is derived from plants and converted to retinol by the liver in approximately a 3 to 1 ratio. Retinol circulates in the blood and is selectively absorbed by cells as it is needed. Within the cells, retinol is converted to one of its two other active forms: retinal or retinoic acid. The rate of conversion is dependent on the vitamin A and protein status of the individual. The 319 Figure 2. Conversion of beta-carotene to retinol (vitamin A, aldehyde). relative amounts of carotenoids directly absorbed and metabolized to vitamin A in the intestine vary greatly . The Recommended Daily Allowance (RDA) for vitamin A is given in retinol equivalents (RE) of active retinol (1 RE 1 mug 3.33 IU). One RE of beta-carotene is 6 mug 10 IU. The adult male RDA for vitamin A is 1000 RE per day and 800 RE per day for an adult female. Vitamin A is found as retinol in foods such as liver, egg yolk, milk, and other dairy products, whereas beta-carotene is found in dark green and yellow vegetables and fruits. Although vitamin A toxicity is not common, it can occur when excess amounts of supplements or major dietary sources, such as liver, are consumed. beta-carotene, on the other hand, is rarely considered toxic, but can accumulate, giving the skin an orange- yellow hue. Role of Vitamin A and Carotenoids in Oxidative Stress Recent studies have demonstrated the antioxidant functions of beta-carotene and carotenoids, in general, and their effective ability to scavenge singlet oxygen.Carotenoids are known to scavenge and deactivate free radicals both in vitro and in vivo. Evidence from in vitro experiments suggests that beta-carotene may exert other antioxidant effects, including inhibition of lipid peroxidation. Whereas some antioxidants prevent the initiation of lipid peroxidation, beta-carotene controls the chain reactions by trapping free radicals, complementing the action of other antioxidant molecules. 320 Carotenoids have the capacity to modulate the enzymatic activities of lipoxygenasesBecause the end products of lipoxygenase activity are proinflammatory and immunomodulatory molecules, there can be many biologic consequences of regulation of the activity of this enzyme by carotenoids. Another carotenoid, lycopene, is less abundant in the diet than beta-carotene. Like beta- carotene, however, it is a singlet oxygen quencher and therefore can act also as an antioxidant. Little data on this carotenoid are presently available. Vitamin A and the Immune Response Depending upon the particular compound, dosage, and method of administration,vitamin A, its precursors, and synthetic derivatives can stimulate or inhibit various aspects of the cellular and humoral immune response. beta-carotene has been shown to stimulate rat lymphocyte mitogenesis in vivo.In vitro studies of the actions of beta-carotene have demonstrated that it increases the numbers of human NK cells and T-helper cells. Animal studies have shown that carotenoids enhance immune function independent of any provitamin A effectCarotenoids have been shown to enhance macrophage function,] thereby modulating the production of reactive oxygen species and controlling actions of other immune cells. Using animal models, Pasatiempo et al have shown that normal antibody function is impaired in early stages of vitamin A deficiency. Bowman et al have shown decreases in NK cell activity and in interferon (IFN) production in the presence of vitamin A deficiency. Much of the research on the effects of carotenoids on human immune responses has been limited to beta-carotene. Watson et al  reported that supplementation of healthy older adults with 30 to 60 mg a day of beta-carotene for 2 months significantly enhanced the percentage of leukocytes in peripheral blood with cell receptors indicative of NK cells and of lymphocytes with interleukin-2 (IL-2) receptors. In that study, plasma levels of beta-carotene, but not of retinol, were significantly elevated, suggesting that modulation of the immune system may have been induced by carotenoid activity rather than by the action of vitamin A.  Vitamin A and Cell Differentiation Vitamin A (as retinol or its activated metabolites) is now recognized as a fundamental factor for growth and cell differentiation. Recent research has shown retinoic acid, an oxidized form of vitamin A, is a key regulator of embryonic development and of the differentiation of epithelial and mesenchymal tissues throughout life.  321 Other Biologic Actions of Vitamin A Vitamin A has essential roles in bone development, the development and maintenance of epithelial tissue, normal reproduction, and vision. By virtue of its effect on protein synthesis and bone cell differentiation, it is necessary for growth and development of skeletal and soft tissues. Vitamin A also plays a role in maintaining normal epithelial structures, including the differentiation of basal cells into mucous epithelial cells.  Vitamin A and Osteoarthritis Although there is great potential for a role of vitamin A in OA, there is virtually no literature that has reported a protective association of dietary or supplemental retinol or beta-carotene in relation to the initiation or progression of OA. VITAMIN C (ASCORBIC ACID) Vitamin C, the antiscorbutic vitamin,  is chemically the least complex of the vitamins. Although plants and many mammals are able to synthesize ascorbic acid from glucose and galactose, humans, monkeys, and guinea pigs do not synthesize vitamin C from intrinsic sources. Ascorbic acid is easily absorbed from the small intestine into the blood by an active mechanism and probably also by diffusion. Average absorption is 90% for intakes between 20 to 120 mg; however, at very high intakes, such as 12 g, which are often self-medicated, absorption is <20% (Fig. 3) . The RDA of 60 mg for adults is based on the amount needed to prevent the onset of scorbutic symptoms for 4 weeks and provide a margin of safety. An increased intake of vitamin C is required to maintain normal plasma or cellular levels in the presence of acute emotional or environmental stress, such as trauma, fever, infection, or elevated environmental temperatures. Sources of dietary vitamin C include citrus fruits and juices, such as oranges and grapefruit, potatoes, tomatoes, broccoli, red peppers, and cooked collard and mustard greens. Figure 3. Vitamin C (reduced and oxidized forms). 322 Role of Vitamin C in Oxidative Stress In human plasma, ascorbate is the only endogenous antioxidant that can completely protect the lipids from detectable peroxidation damage induced by aqueous peroxyl radicals.  Ascorbate is thought to trap virtually all peroxyl radicals in the aqueous phase before they can diffuse into the plasma lipids  ; however, once the ascorbate antioxidative capacity has been expended, the remaining water-soluble antioxidants, urate, bilirubin, and the protein thiols, can trap only part of the aqueous peroxyl radicals. The peroxyl radicals that escape these remaining antioxidants in the aqueous phase diffuse into the plasma lipids, where they initiate lipid peroxidation. Other antioxidants are able to slow the rate at which lipid peroxidation occurs, whereas ascorbate can prevent initiation of detectable lipid peroxidation by aqueous peroxyl radicals.  For this reason, vitamin C has been referred to as the first line of defense  and has been identified as a scavenger of several radicals.  Ascorbate reacts rapidly with both superoxide and peroxyl radicals and even more rapidly with hydroxyl radicals.   It also scavenges singlet oxygen, reduces thiol radicals, and combines rapidly with hypochlorous acid at sites of inflammation. Vitamin C is also believed to have a role in sparing or recycling of alpha-tocopherol (vitamin E)   and has been shown to spare carotenoids  in vitro. It plays a vital role in maintaining the balance between oxidative products and the various cellular antioxidant defense mechanisms. The interdependency of such reactions involves ascorbic acid, vitamin E, selenium, catalase, and glutathione.  It is known that ascorbate can switch from anti- to pro-oxidant activity, depending on its concentration and the presence of free transition metal ions    ; however, in plasma, transition metal ions are tightly bound and unavailable for free radical reactions.     The antioxidant protection afforded by ascorbate does not correlate linearly with its concentration in the presence of unrestricted intake. With increasing concentrations of ascorbate, its efficiency for scavenging free radicals declines. Data from the work of Frei et al  suggest that an increase in the RDA for ascorbate from the current 60 mg to approximately 150 mg (to maximize the total body pool of ascorbate) would be beneficial to human health. Vitamin C and the Immune Response Frei et al  have described plasma ascorbate as being outstandingly effective against the oxidants released from activated polymorphonuclear leukocytes. Vitamin C may promote resistance to infection by altering the immunologic activity of leukocytes, the production of interferon, the inflammatory response, or the integrity of the mucous membranes.  Ascorbate has been shown to have a major influence on phagocyte mobility and chemotaxis.  Before particle ingestion, phagocytes exhibit a marked increase in metabolic activity known as the respiratory burst. Oxygen consumption is greatly increased and much of this extra oxygen 323 is converted to reactive oxygen metabolites. The ROS are released into the phagosome to be used for killing phagocytosed microorganisms; however, ROS are also released to the exterior of the cell, where they may damage adjacent normal tissue.  Data from guinea pigs (which, like humans, do not synthesize vitamin C) have suggested that vitamin C intake is a critical factor in the development of the immune response.  These models confirm the role of vitamin C in phagocyte cell function and suggest effects on cytokine production. Subclinical ascorbate deficiency can be relatively common, even in affluent societies.   Physiologic stress may cause a sharp drop in ascorbate levels, and may occur partly as a result of leukocyte migration to sites of tissue injury.  Increasing dosages of vitamin C in human volunteers were found to increase neutrophil chemotaxis.  Vitamin C may act at more that one level of the immune response, and at least some of these interactions involve specific regulation of the cytokine network. Vitamin C and Osteoarthritis Vitamin C acts as an electron donor in the synthesis of type II collagen, a primary structural component of cartilage. Vitamin C is involved in the hydroxylation of proline to form hydroxyproline in the synthesis of collagen.  It also plays a part in glycosaminoglycan synthesis through its role as a carrier of sulfate groups.  Depletion of sulfated proteoglycans from the extracellular matrix of articular cartilage is one of the earliest manifestations of OA. The higher levels of matrix-degrading enzymes that have been shown to be present in the diseased tissue are thought to play a major role in this loss.  Deficiency of vitamin C, therefore, could be related to the reduced mechanical integrity of the extracellular matrix of articular cartilage and the increased matrix turnover rate in OA.   Schwartz et al  studied the development of surgically induced OA in guinea pigs that were given either high or low (but nonscorbutic) dietary levels of vitamin C. They reported that guinea pigs maintained on high dietary levels of vitamin C developed less- severe OA. The cartilage surfaces in the surgically modified joints showed less loss of cartilage, less pitting and ulceration, and less eburnation in the animals receiving higher dietary levels of vitamin C. In normal articular cartilage, ascorbate significantly increased the biosynthesis of sulfated proteoglycans and inhibited the activity of acid phosphatase.  These properties of vitamin C presumably contributed to the increased formation and stability of proteoglycans that successfully counteracted the erosion of cartilage during development of OA in this animal model. VITAMIN E (alpha-TOCOPHEROL) Dietary vitamin E includes the tocopherols--alpha, beta, gamma, and delta--and the tocotrienols, whose most important chemical characteristic is their 324 antioxidant property. Absorption of vitamin E is relatively inefficient, ranging between 20% and 80%. This vitamin is stored in liver and, to a large extent, in fatty tissues. In food, vitamin E acts to prevent the peroxidation of PUFAs. It enhances the activity of vitamin A by preventing its oxidation in the intestinal tract. At the cellular level, by scavenging free radicals that contain oxygen, vitamin E seems to protect cellular and subcellular membranes from damage. In the absence of vitamin E, free radicals catalyze peroxidation of the PUFAs that constitute structural components of tissue membranes. The requirement for vitamin E depends on the amount of PUFA consumed, which varies widely among individuals. Seed oils, particularly wheat germ oil, are the richest source of vitamin E, although lesser amounts occur in fruits, vegetables, and animal fats. Peanut, olive, coconut, and fish oils are poor sources of the vitamin. Vitamin E as an Antioxidant Vitamin E is a fat-soluble vitamin with a highly flexible structure, facilitating its antioxidant function. The hydrophobic terminal of the vitamin can attach in the lipid bilayer of the cell membrane. The terminal with a hydroxylated aromatic ring can then be located near the water-membrane interface, where it is available to donate an electron to those atoms with unpaired orbits.   Vitamin E has several isomeric structures, but alpha-tocopherol is the isomer with the greatest biologic activity. An electron from the hydroxyl group at the active site can be transferred, converting alpha-tocopherol to its radical form, alpha-tocopheroxyl. Unlike the radical forms of some molecules, the alpha-tocopheroxyl radical is very stable, because of the nature of its ring structure. The propagation of excessive free radicals by vitamin E is controlled by oxidizing alpha-tocopheroxyl to alpha-tocopheryl quinone or by reducing it back to its alpha-tocopherol form in the presence of vitamin C (Fig. 4) .  Vitamin E acts as an antioxidant by preventing lipid peroxidation from entering the chaining process in the propagation phase. Like vitamin C, vitamin E is a sacrificial antioxidant; its antioxidant capacity is therefore expended after it has scavenged a certain number of radicals.  The effects of vitamin E and those of selenium, sulfur amino acids, polyunsaturated fatty acids, and other antioxidants are interdependent.  Figure 4. alpha-Tocopherol. 325 Vitamin E may be beneficial to the body's host defense mechanisms by preventing the infection-induced increase in production of tissue prostaglandins from arachidonic acid.  Prostaglandin production requires an active oxygen species, and lipid peroxides can stimulate synthesis by providing an oxygen species. Goetzl  reported that vitamin E bidirectionally modulated the activity of the lipoxygenase pathway of human neutrophils in vitro and that normal plasma concentrations of vitamin E enhanced the lipoxygenation of arachidonic acid, whereas higher concentrations exerted a suppressive effect, consistent with the role of alpha- tocopherol as a hydroperoxide scavenger. When endothelial cells are exposed to oxidative stress, they synthesize more PGI2 , which may be a protective response against injury.  Meydani et al  have shown that vitamin E supplementation in mice increases lung PGI2 synthesis. Vitamin E and the Immune Response The mounting of an immune response requires membrane-bound, receptor-mediated communication between cells and between protein and lipid mediators. This can be directly or indirectly affected by tocopherol status, although the precise mechanism is not known.  Previous work has shown that free radical and lipid peroxidation reactions play a part in cellular damage.  Evidence from animal models shows that vitamin E is protective against environmental pollutants, decreases thrombosis, reduces the formation of certain carcinogens, and enhances almost every aspect of the immune response. The resistance to infection, specific antibody responses, splenic plaque-forming cells, in vitro mitogenic responses of lymphocytes, reticuloendothelial system clearance, and phagocytosis all are altered by vitamin E deficiency. Increases in tocopherol intake have a positive impact on these processes.  Studies in humans indicate that healthy individuals eating "normal" diets show a significant decrease in markers of cell damage, such as lipid peroxidation and pentane output, when they supplement their diets with vitamin E.   These studies suggest that the average diet may not provide adequate protection against free-radical damage. Vitamin E and Osteoarthritis There have been few studies of vitamin E activity in relation to OA. In a cross-over study, 29 OA patients were randomly assigned to treatment with tocopherol, 600 mg a day for 10 days, or to the placebo group. 326 Machtey et al  found that 52% of those receiving vitamin E experienced marked relief of pain, as compared with only 1 patient (4%) in the placebo group. VITAMIN D The precursors of vitamin D are present in sterol fractions of animal and plant tissues in the form of 7-dehydrocholesterol and ergosterol, respectively. Both precursors require ultraviolet irradiation for conversion to the provitamin form (D3 , cholecalciferol, and D2 ) and both provitamins require conversion in the kidney to the metabolically active form. The plant form is of interest primarily as a food additive (Fig. 5) .  The RDA for vitamin D is 10 mug (400 IU) for children beyond 6 months of age, young adults up to 25 years of age, and pregnant and lactating women. For adults 25 years and older, the RDA is 5 mug. The normal adult is presumed to obtain sufficient vitamin D from exposure to sunlight and the incidental ingestion of small amounts in foods. Because vitamin D is fat soluble, hypervitaminosis D can occur from excess consumption of supplements, including fish oils.  Figure 5. Structural formulae and conversion of provitamin D to vitamin D. 327 Vitamin D and Osteoarthritis Relatively little is known of the direct actions of vitamin D and its metabolites in arthritis. It is likely that vitamin D metabolites are concerned with normal cartilage integrity,  but specific metabolic actions are not well defined. It has been shown that cultured chondrocytes convert vitamin D to its active forms.   Fairney et al  found significant amounts of 25-OH-D and 24, 25-(OH)2 -D in samples of synovial fluid from patients with OA and rheumatoid arthritis (RA). Serum levels of 25- OH-D were, on average, twice as great as those in synovial fluid from the same patient. The investigators suggested that the composition of synovial fluid is consistent with it being a dialysate of plasma into which hyaluronan is secreted by the synovial membrane. Therefore, the presence of vitamin D metabolites in synovial fluid may simply reflect the serum values. In contrast, analyses of 24, 25-(OH)2 -D in samples of synovial fluid and serum suggested local production of that metabolite in the synovium, rather than only diffusion into the joint space from serum. No differences were noted between results of assays of synovial fluid from patients with OA and those with RA. Interest in vitamin D increased when, in 1996, the Framingham Study investigators reported that a relative deficit of vitamin D, as determined by dietary intake and serum levels, predisposes patients to OA of the knee.  They reported that persons with low intake of dietary vitamin D (3-170 IU/day) and lower serum levels of vitamin D (4.9-24.0 ng/mL) were approximately three times more likely to exhibit progression of established knee OA than those having higher intakes of dietary vitamin D (400-1600 IU) and higher levels of serum vitamin D (36.0-79.0 ng/ml). They found no evidence, however, that these lower dietary intakes and lower serum levels influenced the risk for development of OA in a previously normal knee. It is not surprising that vitamin D intakes or serum levels correlate with progression of knee OA, because both are well-known phenomena correlated with the aging process. It is surprising, however, that including measures of bone mineral density in statistical models did not alter the association between vitamin D and OA, especially when the investigators suggested that bone status was a potential mechanism through which vitamin D might influence the progression of OA. Defining whether investigators are describing the initiation or progression of OA is vital to the interpretation of this association between vitamin D and OA. It has been observed for a while now that individuals with OA are more likely to have greater bone mineral density (BMD), and that they had that higher BMD before their presentation with radiographically defined OA. Adequate vitamin D status, either as a result of dietary intake or appropriate sunlight exposure, is typically a component of normal BMD, but has not been described as a component of higher BMD. Thus, it is consistent that the Framingham investigators  found no protection for vitamin D with the development of incident OA. 328 This study, which has stimulated much interest from practitioners and patients, in truth merely highlights the need to study the role of nutrition with a clear sense of the multifactorial nature of OA and the multiple roles that a specific nutrient may have in cellular physiology. Vitamin D, the Immune Response, and Osteoarthritis One possible explanation for the role vitamin D may have played in the progression of OA among the Framingham study subjects lies in vitamin D's potential to contribute to the immunologic response to inflammatory products. Progression of OA associated with a chronic and insidious inflammatory state has not been investigated relative to vitamin D, but studies from the other more inflammatory arthritides may be informative. Peripheral blood lymphocytes and synovial fibroblasts from patients with rheumatoid arthritis possess specific receptors for 1, 25-(OH)2 -D, although the role of the metabolite in these cells is unknown.  Studies of the extrarenal metabolism of 25-(OH)-D show that 1, 25-(OH)2 -D is synthesized in vitro by normal human macrophages activated with interferon gamma  or bacterial lipopolysaccharides.  Hayes et al  have speculated that activated macrophages in patients with arthritis synthesize 1, 25-(OH)2 -D within the affected joints. These investigators examined synovial fluid samples from 45 patients with knee effusions and showed that synovial fluid cells were capable of synthesizing the active vitamin D metabolite 1, 25-(OH)2 -D and that activated macrophages appeared to be responsible for much of the 1, 25(OH)2 -D synthesized. Although the significance of the ability of macrophages from patients with inflammatory arthritis to synthesize 1, 25- (OH)2 -D is not clear, Hayes et al  speculate that the importance may be greater when the cells are present within the synovial membrane, and thus adjacent to the sites of tissue damage and bone erosion within the joint, than when they are present in the synovial fluid. Overall, specific receptors for 1, 25-(OH)2 -D on normal human monocytes, activated lymphocytes, and peripheral blood lymphocytes and synovial fibroblasts from patients with rheumatoid arthritis  suggest a physiologic role for the metabolite in joint disease. Many of the reported effects of 1, 25-(OH)2 -D may be relevant to OA. For example, it appears to inhibit proliferation of B and T lymphocytes and to reduce interleukin-2 production by activated T lymphocytes in vitro.  Although synthesis of 24, 25-(OH)2 D has been demonstrated in normal human macrophages  and in articular cartilage and chondrocyte cultures,  Hayes et al did not find evidence of synthesis of 24, 25-(OH)2 D in synovial fluid cells of the 45 patients with knee effusions described previously.  Vitamin D, Cell Differentiation, Bone, and Immunologic Responsiveness Vitamin D plays multiple roles that ultimately may have an effect in OA. The hormonal form of vitamin D (1, 25-(OH)2 -D) inhibits collagen 329 synthesis by osteoblasts and promotes bone resorption. Hormonal vitamin D therefore may contribute to OA status insofar as OA is determined by bone mineral density. In addition, 1, 25-(OH)2 -D promotes differentiation of monocytes into macrophages and the fusion of macrophages into multinucleated giant cells. These giant cells can have multiple functions, including a humoral immunological response and stimulation of bone resorbing activity by osteoclasts.  1, 25-(OH)2 -D also acts upon T lymphocytes, producing a variety of lymphokines, including the potent bone resorbing agent osteoclast activating factor.  Within the synovial fluid and synovium 1, 25-(OH)2 -D thus may have complex paracrine and immunoregulatory functions.  It is also important to note that these roles are played by the hormonal form of vitamin D, a form that is only indirectly associated with measures of dietary vitamin D intake, sunlight exposure, and serum levels of 25-hydroxyvitamin D.