n⫺3 Polyunsaturated fatty acids, inflammation, and inflammatory
`diseases1–3
`
`Downloaded from
`
`ajcn.nutrition.org
`
` by guest on October 12, 2017
`
`endothelial cells, which allows leukocyte binding and subse-
`quent diapedesis. The earliest cells to appear at inflamed sites are
`granulocytes, with monocytes, macrophages, and lymphocytes
`appearing later. Granulocytes, monocytes, and macrophages are
`involved in pathogen killing, in clearing up cellular and tissue
`debris, and in tissue repair. The activity of these cells is induced
`by certain triggers. One important exogenous trigger is bacterial
`endotoxin (also known as lipopolysaccharide), a component of
`the cell wall of Gram-negative bacteria, which can directly ac-
`tivate monocytes and macrophages, inducing them to form cy-
`tokines, such as tumor necrosis factor ␣(TNF-␣); interleukin 1
`(IL-1), IL-6, and IL-8; eicosanoids, such as prostaglandin (PG)
`E2; nitric oxide; matrix metalloproteinases; and other mediators.
`Endotoxin also induces adhesion molecule expression on the
`surface of endothelial cells and leukocytes.
`The cytokines produced by monocytes and macrophages also
`serve to regulate the whole-body response to infection and injury
`(Figure 1). Thus, inflammation and the inflammatory response
`are part of the normal, innate immune response. Inflammatory
`mediators also provide a link between innate and acquired im-
`mune responses (Figure 1). The actions of inflammatory cyto-
`kines, which initiate a cascade of inflammatory mediators, thus
`amplifying the initial inflammatory signal, are opposed by anti-
`inflammatory cytokines such as IL-10 and by receptor antago-
`nists such as IL-1 receptor antagonist.
`Although inflammation is a normal response, when it occurs in
`an uncontrolled or inappropriate manner, excessive damage to
`host tissues and disease can ensue. Such uncontrolled or inap-
`propriate inflammatory responses are characterized by hyper-
`expression of endothelial and leukocyte adhesion molecules,
`appearance of soluble forms of adhesion molecules in the circu-
`lation, sequestration of leukocytes to sites where they are not
`usually found, production of inflammatory mediators, and dam-
`age to host tissues (Figure 2). High concentrations of TNF-␣,
`IL-1␤, and IL-6 are particularly destructive and are implicated in
`some of the pathologic responses that occur in endotoxic shock,
`in acute respiratory distress syndrome, and in chronic inflamma-
`tory diseases such as rheumatoid arthritis and inflammatory
`
`1 From the Institute of Human Nutrition, School of Medicine, University
`of Southampton, Southampton, United Kingdom.
`2 Presented at the symposium “nҀ3 Fatty Acids: Recommendations for
`Therapeutics and Prevention,” held at the Institute of Human Nutrition,
`Columbia University, New York, NY, 21 May 2005.
`3 Address reprint requests to PC Calder, Institute of Human Nutrition,
`School of Medicine, University of Southampton, Bassett Crescent East,
`Southampton SO16 7PX, United Kingdom. E-mail: pcc@soton.ac.uk.
`
`Philip C Calder
`
`ABSTRACT
`Inflammation is part of the normal host response to infection and
`injury. However, excessive or inappropriate inflammation contrib-
`utes to a range of acute and chronic human diseases and is charac-
`terized by the production of inflammatory cytokines, arachidonic
`acid– derived eicosanoids (prostaglandins, thromboxanes, leukotri-
`enes, and other oxidized derivatives), other inflammatory agents (eg,
`reactive oxygen species), and adhesion molecules. At sufficiently
`high intakes, long-chain nҀ3 polyunsaturated fatty acids (PUFAs),
`as found in oily fish and fish oils, decrease the production of inflam-
`matory eicosanoids, cytokines, and reactive oxygen species and the
`expression of adhesion molecules. Long-chain nҀ3 PUFAs act both
`directly (eg, by replacing arachidonic acid as an eicosanoid substrate
`and inhibiting arachidonic acid metabolism) and indirectly (eg, by
`altering the expression of inflammatory genes through effects on
`transcription factor activation). Long-chain nҀ3 PUFAs also give
`rise to a family of antiinflammatory mediators termed resolvins.
`Thus, nҀ3 PUFAs are potentially potent antiinflammatory agents.
`As such, they may be of therapeutic use in a variety of acute and
`chronic inflammatory settings. Evidence of their clinical efficacy is
`reasonably strong in some settings (eg, in rheumatoid arthritis) but is
`weak in others (eg, in inflammatory bowel diseases and asthma).
`More, better designed, and larger trials are required to assess the
`therapeutic potential of long-chain nҀ3 PUFAs in inflammatory
`diseases. The precursor nҀ3 PUFA ␣-linolenic acid does not appear
`to exert antiinflammatory effects at achievable intakes.
`Am J
`Clin Nutr 2006;83(suppl):1505S–19S.
`
`KEY WORDS
`Inflammation, monocyte, macrophage, eico-
`sanoid, cytokine, inflammatory disease
`
`INFLAMMATION IN HEALTH AND DISEASE
`Inflammation is part of the body’s immediate response to
`infection or injury. It is typified by redness, swelling, heat, and
`pain. These occur as a result of increased blood flow; increased
`permeability across blood capillaries, which permits large mol-
`ecules (eg, complement, antibodies, and cytokines) to leave the
`bloodstream and cross the endothelial wall; and increased move-
`ment of leukocytes from the bloodstream into the surrounding
`tissue. Inflammation functions to begin the immunologic process
`of elimination of invading pathogens and toxins and to repair
`damaged tissue. These responses must be ordered and controlled.
`The movement of cells into the inflammatory or infected site is
`induced by the up-regulation of adhesion molecules such as
`intercellular adhesion molecule 1 (ICAM-1), vascular cell adhe-
`sion molecule 1 (VCAM-1), and E-selectin on the surface of
`
`Am J Clin Nutr 2006;83(suppl):1505S–19S. Printed in USA. © 2006 American Society for Nutrition
`
`1505S
`
`AKER EXHIBIT 2005 Page 1
`
`

`

`1506S
`
`CALDER
`
`Downloaded from
`
`ajcn.nutrition.org
`
` by guest on October 12, 2017
`
`FIGURE 1. The role of inflammatory cells and mediators in regulating the whole-body metabolic and immunologic responses to infection and injury.
`Modified from reference 1 with permission from the American Oil Chemists’ Society.
`
`bowel disease. Chronic overproduction of TNF-␣and IL-1 can
`cause adipose tissue and muscle wasting and loss of bone mass
`and may account for alterations in body composition and tissue
`loss seen in inflammatory diseases and in cancer cachexia. As
`well as its clear and obvious association with classic inflamma-
`tory diseases, inflammation is now recognized to play an impor-
`tant role in the pathology of other diseases, such as cardiovas-
`cular disease and neurodegenerative diseases of aging.
`Additionally, the realization that adipose tissue is a source of
`inflammatory cytokines has given rise to the notion that obesity,
`the metabolic syndrome, and type 2 diabetes have an inflamma-
`tory component.
`
`FIGURE 2. Diagrammatic representation of the movement of leukocytes
`through the endothelium and the subsequent generation of inflammatory
`mediators.
`
`ARACHIDONIC ACID–DERIVED EICOSANOIDS AND
`INFLAMMATION
`The key link between polyunsaturated fatty acids (PUFAs)
`and inflammation is that eicosanoids, which are among the me-
`diators and regulators of inflammation, are generated from 20-
`carbon PUFAs. Because inflammatory cells typically contain a
`high proportion of the nҀ6 PUFA arachidonic acid (20:4nҀ6)
`and low proportions of other 20-carbon PUFAs, arachidonic acid
`is usually the major substrate for eicosanoid synthesis. Eico-
`sanoids, which include PGs, thromboxanes, leukotrienes (LTs),
`and other oxidized derivatives, are generated from arachidonic
`acid by the metabolic processes summarized in Figure 3. Eico-
`sanoids are involved in modulating the intensity and duration of
`inflammatory responses (see references 2 and 3 for reviews),
`have cell- and stimulus-specific sources, and frequently have
`opposing effects (Table 1). Thus, the overall physiologic (or
`pathophysiologic) outcome will depend on the cells present, the
`nature of the stimulus, the timing of eicosanoid generation, the
`concentrations of different eicosanoids generated, and the sen-
`sitivity of the target cells and tissues to the eicosanoids generated.
`Recent studies have shown that PGE2 induces cyclooxygenase 2
`(COX-2) in fibroblasts cells and so up-regulates its own produc-
`tion (5), induces the production of IL-6 by macrophages (5),
`inhibits 5-lipoxygenase (5-LOX) and so decreases production of
`the 4-series LTs (6), and induces 15-LOX and so promotes the
`formation of lipoxins (6, 7), which have been found to have
`antiinflammatory effects (8, 9). Thus, PGE2 possesses both pro-
`and antiinflammatory actions (Table 1).
`
`AKER EXHIBIT 2005 Page 2
`
`

`

`nҀ3 PUFAs AND INFLAMMATION
`
`1507S
`
`Downloaded from
`
`ajcn.nutrition.org
`
` by guest on October 12, 2017
`
`FIGURE 3. Generalized pathway for the conversion of arachidonic acid to eicosanoids. COX, cyclooxygenase; HETE, hydroxyeicosatetraenoic acid;
`HPETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; LT, leukotriene; PG, prostaglandin; TX, thromboxane.
`
`ARACHIDONIC ACID AND INFLAMMATORY
`MEDIATOR PRODUCTION
`Animal feeding studies have shown a strong positive relation
`between the amount of arachidonic acid in inflammatory cells
`and the ability of those cells to produce eicosanoids such as PGE2
`(10). In turn, the amount of arachidonic acid in inflammatory
`cells can be increased by including arachidonic acid in the diet of
`rats (10) or by increasing the amount of it in the diet of humans
`
`TABLE 1
`Pro- and antiinflammatory effects of prostaglandin E2 (PGE2) and
`leukotriene B4 (LTB4)1
`
`PGE2
`Proinflammatory
`Induces fever
`Increases vascular permeability
`Increases vasodilatation
`Causes pain
`Enhances pain caused by other agents
`Increases production of IL-6
`Antiinflammatory
`Inhibits production of TNF and IL-1
`Inhibits 5-LOX (decreases 4-series LT production)
`Induces 15-LOX (increases lipoxin production)
`LTB4
`Proinflammatory
`Increases vascular permeability
`Enhances local blood flow
`Chemotactic agent for leukocytes
`Induces release of lysosomal enzymes
`Induces release of reactive oxygen species by granulocytes
`Increases production of TNF, IL-1, and IL-6
`
`1 IL, interleukin; LOX, lipoxygenase; TNF, tumor necrosis factor. Mod-
`ified from reference 4 with permission from the American Oil Chemists’
`Society.
`
`(11). The amount of arachidonic acid in inflammatory cells
`may also be influenced by dietary intake of its precursor,
`linoleic acid (18:2nҀ6), although the range of linoleic acid
`intake over which this relation occurs has not been defined for
`humans. Increasing linoleic acid intake by 6.5 g/d in humans
`who habitually consume 10 –15 g/d did not alter the arachi-
`donic acid content of blood mononuclear cells (12). Never-
`theless, the role of arachidonic acid as a precursor for the
`synthesis of eicosanoids indicates the potential for dietary
`nҀ6 PUFAs (linoleic or arachidonic acid) to influence in-
`flammatory processes. This has been little investigated in
`humans. Supplementation of the diet of healthy young men
`with 1.5 g arachidonic acid/d for 7 wk resulted in a marked
`increase in production of PGE2 and LTB4 by endotoxin-
`stimulated mononuclear cells (13). However, production of
`TNF-␣, IL-1␤, and IL-6 by these cells was not significantly
`altered (13). Thus, increased arachidonic acid intake may
`result in changes indicative of selectively increased inflam-
`mation or inflammatory responses in humans. Supplementa-
`tion of the diet of healthy elderly subjects with arachidonic
`acid [0.7 g/d in addition to a habitual intake of 앒0.15 g/d (11)]
`for 12 wk did not affect endotoxin-stimulated production of
`TNF-␣, IL-1␤, or IL-6 by mononuclear cells; did not alter
`reactive oxygen species (superoxide) production by neutro-
`phils or monocytes; and did not alter plasma soluble
`VCAM-1, ICAM-1, or E-selectin concentrations (14). This
`lack of effect was despite incorporation of arachidonic acid
`into target cells (11). Taken together, these studies suggest
`that modestly increased intake of arachidonic acid results in
`incorporation of arachidonic acid into cells involved in in-
`flammatory responses (11), but that this does not affect the
`production of inflammatory cytokines (13, 14), the generation
`of superoxide (14), or the shedding of adhesion molecules
`(14), although production of inflammatory eicosanoids is in-
`creased (13).
`
`AKER EXHIBIT 2005 Page 3
`
`

`

`1508S
`
`CALDER
`
`Downloaded from
`
`ajcn.nutrition.org
`
` by guest on October 12, 2017
`
`EPA can also act as a substrate for both COX and 5-LOX, giving
`rise to eicosanoids with a slightly different structure from those
`formed from arachidonic acid (Figure 5). Thus, fish oil supplemen-
`tation of the human diet has been shown to result in increased pro-
`duction of LTB5, LTE5, and 5-hydroxyeicosapentaenoic acid by
`inflammatory cells (15, 17, 23), although generation of PGE3 has
`been more difficult to demonstrate (25). The functional significance
`of this is that the mediators formed from EPA are believed to be less
`potent than those formed from arachidonic acid. For example, LTB5
`is 10- to 100-fold less potent as a neutrophil chemotactic agent than
`LTB4 (26, 27). Recent studies have compared the effects of PGE2
`and PGE3 on production of cytokines by cell lines and by human
`cells. Bagga et al (5) reported that PGE3 was a less potent inducer of
`COX-2 gene expression in fibroblasts and of IL-6 production by
`macrophages. However, PGE2 and PGE3 had equivalent inhibitory
`effects on the production of TNF-␣ (28, 29) and IL-1␤ (29) by
`human mononuclear cells stimulated with endotoxin. The reduction
`in generation of arachidonic acid–derived mediators that accompa-
`nies fish oil consumption has led to the idea that fish oil is antiin-
`flammatory (Figure 6).
`In addition to long-chain nҀ3 PUFAs modulating the gener-
`ation of eicosanoids from arachidonic acid and to EPA acting as
`a substrate for the generation of alternative eicosanoids, recent
`studies have identified a novel group of mediators, termed
`E-series resolvins, formed from EPA by COX-2 that appear to
`exert antiinflammatory actions (30 –32). In addition, DHA-
`derived mediators termed D-series resolvins, docosatrienes and
`neuroprotectins, also produced by COX-2, have been identified
`and also appear to be antiinflammatory (33–35). This is an ex-
`citing new area of nҀ3 fatty acids and inflammatory mediators
`and the implications for a variety of conditions may be of great
`importance. This area was recently reviewed (36, 37).
`
`ANTIINFLAMMATORY EFFECTS OF LONG-CHAIN
`nⴚ3 PUFAs OTHER THAN ALTERED EICOSANOID
`PRODUCTION
`Although their action in antagonizing arachidonic acid metab-
`olism is a key antiinflammatory effect of nҀ3 PUFAs, these fatty
`
`FIGURE 4. Relation between tuna oil consumption and the fatty acid
`content of human neutrophils. Healthy male volunteers consumed differing
`amounts of tuna oil in capsules for 12 wk. Neutrophils were isolated before
`and at the end of the intervention period, and the fatty acid composition of
`their phospholipids determined. The mean changes in the proportions of
`arachidonic acid, docosahexaenoic acid (DHA), and eicosapentaenoic acid
`(EPA) were linearly related to the increase in tuna oil consumption (g/d). Data
`are from reference 20.
`
`LONG-CHAIN nⴚ3 PUFAs AND INFLAMMATORY
`EICOSANOID PRODUCTION
`Increased consumption of long-chain nҀ3 PUFAs, such as
`eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid
`(DHA; 22:6n-3), results in increased proportions of those fatty
`acids in inflammatory cell phospholipids (12, 15–20). The in-
`corporation of EPA and DHA into human inflammatory cells
`occurs in a dose-response fashion and is partly at the expense of
`arachidonic acid (Figure 4). Because less substrate is available
`for synthesis of eicosanoids from arachidonic acid, fish oil sup-
`plementation of the human diet has been shown to result in
`decreased production of PGE2 (16, 19, 21, 22), thromboxane B2
`(19), LTB4 (15, 17), 5-hydroxyeicosatetraenoic acid (15, 17),
`and LTE4 (23) by inflammatory cells. Although these studies
`used fish oil, Kelley et al (24) showed that 6 g DHA/d resulted in
`decreased production of PGE2 (by 60%) and LTB4 (by 75%) by
`endotoxin-stimulated mononuclear cells.
`
`FIGURE 5. Generalized pathway for the conversion of eicosapentaenoic acid to eicosanoids. COX, cyclooxygenase; HEPE, hydroxyeicosapentaenoic acid;
`HPEPE, hydroperoxyeicosapentaenoic acid; LOX, lipoxygenase; LT, leukotriene; PG, prostaglandin; TX, thromboxane.
`
`AKER EXHIBIT 2005 Page 4
`
`

`

`nҀ3 PUFAs AND INFLAMMATION
`
`1509S
`
`Downloaded from
`
`ajcn.nutrition.org
`
` by guest on October 12, 2017
`
`dose-response study by Schmidt et al (41) suggests that near-
`maximum inhibition of chemotaxis occurs at an intake of 1.3 g
`EPAѿDHA/d. A lower intake (0.55 g EPAѿDHA/d) did not
`affect monocyte chemotaxis (42). However, Healy et al (20) did
`not find an effect of several doses of fish oil providing up to 2.25 g
`EPAѿDHA/d on neutrophil chemotaxis. The apparently diver-
`gent reports of Schmidt et al (42) and Healy et al (20) could be
`explained by the fact that the latter study used a low-EPA, high-
`DHA fish oil such that the highest dose provided 0.58 g EPA/d,
`which is less than the amount of EPA provided by the lowest dose
`of fish oil used by Schmidt et al. If this is so, then the anti-
`chemotactic effects of fish oil might be due to EPA rather than
`DHA. No studies have attempted to discriminate the effects of
`EPA and DHA on chemotaxis.
`
`Long-chain nⴚ3 PUFAs and adhesion molecule
`expression
`Cell culture (43– 46) and animal feeding studies (47) report
`decreased expression of some adhesion molecules on the surface
`of monocytes (46), macrophages (47), or endothelial cells (43–
`45) after exposure to long-chain nҀ3 PUFAs. Supplementing the
`diet of healthy humans with fish oil providing 앒1.5 g
`EPAѿDHA/d results in a lower level of expression of ICAM-1
`on the surface of blood monocytes stimulated ex vivo with
`interferon-␥(48). Dietary fish oil providing 1.1 g EPAѿDHA/d
`was found to decrease circulating concentrations of soluble
`VCAM-1 in elderly subjects (49), but it is not clear whether this
`represents decreased surface expression of VCAM-1.
`
`Long-chain nⴚ3 PUFAs and reactive oxygen species
`production
`Supplementation studies providing 3.1– 8.4 g EPAѿDHA/d
`have reported 30 –55% decreases in the production of reactive
`oxygen species (superoxide or hydrogen peroxide) by stimulated
`human neutrophils
`(50 –52). Supplementation with 6 g
`EPAѿDHA/d was shown to decrease hydrogen peroxide pro-
`duction by human monocytes (53). Studies using lower doses of
`long-chain nҀ3 PUFAs (0.55–2.3 g/d) failed to demonstrate
`
`FIGURE 6. Classic mechanism of the antiinflammatory action of long-
`chain nҀ3 fatty acids. Eicosapentaenoic acid (EPA) and docosahexaenoic
`acid (DHA) decrease the amounts of arachidonic acid available as a substrate
`for eicosanoid synthesis and also inhibit the metabolism of arachidonic acid.
`COX, cyclooxygenase; LOX, lipoxygenase; LT, leukotriene; PG, prosta-
`glandin; TX, thromboxane.
`
`acids have several other antiinflammatory effects that might re-
`sult from altered eicosanoid production or might be independent
`of this. For example, studies have shown that, when consumed in
`sufficient quantities, dietary fish oil results in decreased leuko-
`cyte chemotaxis, decreased production of reactive oxygen spe-
`cies and proinflammatory cytokines, and decreased adhesion
`molecule expression (Table 2).
`
`Long-chain nⴚ3 PUFAs and leukocyte chemotaxis
`Several dietary supplementation studies that used between 3.1
`and 14.4 g EPAѿDHA/d have shown a time-dependent decrease
`in chemotaxis of human neutrophils and monocytes toward var-
`ious chemoattractants, including LTB4, bacterial peptides, and
`human serum (15–17, 38 – 40). Both the distance of cell migra-
`tion and the number of cells migrating were decreased. Despite
`the high dose of long-chain nҀ3 PUFAs used in these studies, a
`
`TABLE 2
`Summary of the antiinflammatory effects of long-chain nҀ3 fatty acids1
`
`Antiinflammatory effect
`Decreased generation of arachidonic acid–derived
`eicosanoids (many with inflammatory actions)
`Increased generation of EPA-derived eicosanoids
`(many with less inflammatory actions than
`those produced from arachidonic acid)
`Increased generation of EPA and DHA-derived
`resolvins (with antiinflammatory actions)
`Decreased generation of inflammatory cytokines
`(TNF-␣, IL-1␤, IL-6, and IL-8)
`
`Decreased expression of adhesion molecules
`
`Decreased leukocyte chemotaxis
`Decreased generation of reactive oxygen species
`
`Mechanism likely to be involved
`Decreased arachidonic acid in cell membrane phospholipids; inhibition of arachidonic acid
`metabolism; decreased induction of COX-2, 5-LOX, and 5-LOX activating protein
`Increased content of EPA in cell membrane phospholipids
`
`Increased content of EPA and DHA in cell membrane phospholipids
`
`Decreased activation of NF␬B (via decreased phosphorylation of I␬B); activation of PPAR␥;
`altered activity of other transcription factors; differential effects of arachidonic acid– vs EPA-
`derived eicosanoids
`Decreased activation of NF␬B (via decreased phosphorylation of I␬B); altered activity of other
`transcription factors
`Not clear; perhaps decreased expression of receptors for some chemoattractants
`Not clear; perhaps altered membrane composition affecting signaling processes
`
`1 COX, cyclooxygenase; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; I␬B, inhibitory subunit of NF␬B; IL, interleukin; LOX, lipoxygenase;
`NF␬B, nuclear factor ␬B; PPAR, peroxisome proliferator-activated receptor; TNF, tumor necrosis factor. Modified from reference 4 with permission from the
`American Oil Chemists’ Society.
`
`AKER EXHIBIT 2005 Page 5
`
`

`

`1510S
`
`CALDER
`
`Downloaded from
`
`ajcn.nutrition.org
`
` by guest on October 12, 2017
`
`FIGURE 7. Inverse relation between the eicosapentaenoic acid (EPA)
`content of human mononuclear cells and the production of tumor necrosis
`factor ␣(TNF␣) and interleukin 1␤(IL-1␤). Healthy male volunteers con-
`sumed various combinations of sunflower oil, flaxseed oil, and fish oil re-
`sulting in various EPA levels in blood mononuclear cells. The mononuclear
`cells were isolated and stimulated ex vivo with endotoxin for 24 h. The total
`intra- and extracellular accumulation of TNF-␣and IL-1␤was measured by
`specific enzyme-linked immunosorbent assays. The concentration of each
`inflammatory cytokine was inversely related to the EPA content of the mono-
`nuclear cells. Reproduced from reference 19 with permission.
`
`and cytokines. The roles of nҀ6 and nҀ3 PUFAs in shaping and
`regulating inflammatory processes and responses suggest that
`the balance of these fatty acids might be important in determining
`the development and severity of inflammatory diseases. For ex-
`ample, a high intake of nҀ6 PUFAs, especially arachidonic acid,
`could contribute to inflammatory processes and so could predis-
`pose to or exacerbate inflammatory diseases. Conversely, the
`recognition that the long-chain nҀ3 PUFAs have antiinflamma-
`tory actions suggests that increasing their intake by patients with
`inflammatory diseases, for example, through dietary supplemen-
`tation, may be of clinical benefit. Possible therapeutic targets for
`long-chain nҀ3 PUFAs are listed in Table 3. Supplementation
`trials have been conducted for most of these diseases. Those trials
`dealing with rheumatoid arthritis, inflammatory bowel diseases
`(Crohn disease and ulcerative colitis), and asthma will be re-
`viewed in some detail here. This is because a larger number of
`trials have been conducted for these diseases or because the
`evidence of benefit is strongest in these diseases.
`
`effects on reactive oxygen species production by either neutro-
`phils (14, 20, 54, 55) or monocytes (14, 42, 54, 55). A study by
`Halvorsen et al (56) reported that 3.8 g of either EPA or DHA per
`day did not affect production of hydrogen peroxide by human
`monocytes. This lack of effect might relate either to the different
`stimulus used in this study (Escherichia coli) compared with the
`other “high” dose study with monocytes (latex beads) (53) or to
`the fact that 3.8 g long-chain nҀ3 PUFAs/d is below and 6 g/d is
`above the threshold that affects hydrogen peroxide production by
`monocytes.
`
`Long-chain nⴚ3 PUFAs and inflammatory cytokine
`production
`Cell culture studies show that EPA and DHA can inhibit the
`production of IL-1␤and TNF-␣by monocytes (57– 60) and the
`production of IL-6 and IL-8 by venous endothelial cells (43, 61).
`Fish oil feeding decreases the ex vivo production of TNF-␣,
`IL-1␤, and IL-6 by rodent macrophages (62– 64). Supplementa-
`tion of the diet of healthy humans with fish oil providing 쏜2 g
`EPAѿDHA/d was shown to decrease the production of TNF or
`IL-1 or IL-6 by mononuclear cells in some studies (16, 19, 21,
`65– 67). Caughey et al (19) reported a significant inverse corre-
`lation between the EPA content of mononuclear cells and the
`ability of those cells to produce TNF-␣and IL-1␤in response to
`endotoxin (Figure 7). Kelley et al (24) showed that 6 g DHA/d
`for 12 wk resulted in decreased production of TNF-␣(by 20%)
`and IL-1␤(by 35%) by endotoxin-stimulated mononuclear cells.
`Thus, although most studies have used fish oil, it appears that
`both EPA (19) and DHA (24) can decrease inflammatory cyto-
`kine production. This is confirmed by a study in which persons
`with type 2 diabetes were given 4 g EPA or DHA/d for 6 wk (68).
`Both EPA and DHA resulted in decreased plasma TNF-␣ con-
`centrations, although DHA was more potent (35% reduction
`compared with 20% for EPA). Note, however, that several other
`studies failed to show effects of dietary long-chain nҀ3 PUFAs
`on the production of inflammatory cytokines in humans. Some of
`these studies provided 쏝2 g EPAѿDHA/d (14, 42, 54, 69, 70),
`although others provided higher doses (12, 55, 71–74). It is not
`clear what the reason for these discrepancies in the literature is,
`but technical factors are likely to contribute (75). The relative
`contributions of EPA and DHA might also be important in de-
`termining the effect of fish oil. One other factor that was recently
`identified is polymorphisms in genes affecting cytokine produc-
`tion (76). It was found that the effect of dietary fish oil on cyto-
`kine production by human mononuclear cells was dependent on
`the nature of the Ҁ308 TNF-␣ and the ѿ252 TNF-␤ polymor-
`phisms. This study raises the possibility of being able to identify
`those who are more likely and those who are less likely to expe-
`rience specific antiinflammatory effects of fish oil.
`
`CLINICAL APPLICATIONS OF THE
`ANTIINFLAMMATORY EFFECTS OF LONG-CHAIN
`nⴚ3 PUFAs
`
`Introductory comments
`Inflammation is an overt or covert component of numerous
`human conditions and diseases. Although the inflammation may
`afflict different body compartments, one common characteristic
`of these conditions and diseases is excessive or inappropriate
`production of inflammatory mediators, including eicosanoids
`
`AKER EXHIBIT 2005 Page 6
`
`

`

`Downloaded from
`
`ajcn.nutrition.org
`
` by guest on October 12, 2017
`
`nҀ3 PUFAs AND INFLAMMATION
`
`1511S
`
`TABLE 3
`Diseases and conditions with an inflammatory component in which long-
`chain nҀ3 fatty acids might be of benefit1
`
`Disease
`
`Rheumatoid arthritis
`Crohn disease
`Ulcerative colitis
`Lupus
`Type 1 diabetes
`Type 2 diabetes
`Cystic fibrosis
`Childhood asthma
`Adult asthma
`Allergic disease
`Psoriasis
`Multiple sclerosis
`Neurodegenerative disease of aging
`Atherosclerosis
`Acute cardiovascular events
`Obesity
`Systemic inflammatory response to surgery, trauma,
`and critical illness
`Acute respiratory distress syndrome
`Cancer cachexia
`
`1 Note: the list is not exhaustive.
`
`Reference to the role
`of inflammation
`
`(77)
`(78)
`(78)
`(79)
`(80)
`(80, 81)
`(82)
`(83)
`(84)
`(85)
`(86)
`(87)
`(87, 88)
`(89, 90)
`(90, 91)
`(92)
`(93)
`
`(94)
`(95)
`
`Rheumatoid arthritis
`Rheumatoid arthritis is a chronic inflammatory disease char-
`acterized by joint inflammation that manifests as swelling, pain,
`functional impairment, morning stiffness, osteoporosis, and
`muscle wasting. Joint lesions are characterized by infiltration of
`activated macrophages, T lymphocytes, and plasma cells into the
`synovium (the tissue lining the joints) and by proliferation of
`synovial cells called synoviocytes. Synovial biopsies from pa-
`tients with rheumatoid arthritis contain high concentrations of
`TNF-␣, IL-1␤, IL-6, IL-8, and granulocyte-macrophage colony-
`stimulating factor (GM-CSF), and synovial cells cultured ex vivo
`produce TNF-␣, IL-1␤, IL-6, IL-8, and GM-CSF for extended
`periods of time without additional stimulus (77). COX-2 expres-
`sion is increased in the synovium of rheumatoid arthritis patients,
`and in the joint tissues in rat models of arthritis (96). PGE2, LTB4,
`5-hydroxyeicosatetraenoic acid, and platelet-activating factor
`are found in the synovial fluid of patients with active rheumatoid
`arthritis (97). The efficacy of nonsteroidal antiinflammatory
`drugs in rheumatoid arthritis indicates the importance of proin-
`flammatory COX pathway products in the pathophysiology of
`the disease. Increased expression of E-selectin, VCAM-1, and
`ICAM-1 is found in patients with arthritis, and blocking ICAM-1
`or VCAM-1 with antibodies reduces leukocyte infiltration into
`the synovium and synovial inflammation in animal models of the
`disease (see 98 for references).
`Dietary fish oil has been shown to have beneficial effects in
`animal models of arthritis. For example, compared with feeding
`vegetable oil, feeding mice fish oil delayed the onset (mean: 34 d
`compared with 25 d) and reduced the incidence (69% compared
`with 93%) and severity (mean peak severity score: 6.7 compared
`with 9.8) of type II collagen-induced arthritis (99). Both EPA and
`DHA suppressed streptococcal cell wall–induced arthritis in rats,
`but EPA was more effective (100).
`
`Several studies have reported antiinflammatory effects of fish
`oil in patients with rheumatoid arthritis, such as decreased LTB4
`production by neutrophils (101–104) and monocytes (103, 105),
`decreased IL-1 production by monocytes (106), decreased
`plasma IL-1␤concentrations (107), decreased serum C-reactive
`protein concentrations (101), and normalization of the neutrophil
`chemotactic response (108). Several randomized, placebo-
`controlled, double-blind studies of fish oil in rheumatoid arthritis
`have been reported. The characteristics and findings of these
`trials are summarized in Table 4. The dose of long-chain nҀ3
`PUFAs used in these trials was between 1.6 and 7.1 g/d and
`averaged 앒3.5 g/d. Almost all of these trials showed some ben-
`efit of fish oil. Such benefits included reduced duration of morn-
`ing stiffness, reduced number of tender or swollen joints, reduced
`joint pain, reduced time to fatigue, increased grip strength, and
`decreased use of nonsteroidal antiinflammatory drugs (Table 4).
`Arachidonic acid may contribute to inflammatory processes
`by acting as a precursor to eicosanoids known to have a role in
`rheumatoid arthritis (77, 78). Additionally, long-chain nҀ3
`PUFAs may act as antiinflammatory agents by competing with
`arachidonic acid for incorporation into inflammatory cell mem-
`branes and for metabolism by enzymes of eicosanoid synthesis.
`Thus, it is possible that greater efficacy of nҀ3 PUFAs may be
`achieved in rheumatoid arthritis by simultaneously decreasing
`nҀ6 PUFA intake, especially that of arachidonic acid. This was
`investigated by Adam et al (116). Fish oil, providing 4.2 g EPA
`ѿ DHA/d, or placebo was given to patients against a background
`of a typical Western diet, providing 0.1– 0.25 g arachidonic ac-
`id/d, or of a diet that restricted the intake of arachidonic acid–rich
`foods (meat, egg yolk, etc) and that provided 0.025– 0.09 g ara-
`chidonic acid/d; the latter diet was termed an antiinflammatory
`diet. The fish oil–induced decreases in plasma concentrations of
`thromboxane A2 and LTB4 and the urinary concentration of PG
`metabolites were greater in patients consuming the antiinflam-
`matory diet than in those consuming the Western diet. The re-
`ductions in the number of swollen joints, number of tender joints,
`patient’s global assessment, physician’s global assessment, and
`patient’s assessment of pain seen with fish oil supplementation
`were all also greater for patients consuming the antiinflammatory
`diet (116). Nonsteroidal antiinflammatory drug use declined in
`patients receiving fish oil against the background of the antiin-
`flammatory diet but not against the background of the Western
`diet.
`Several reviews of the trials of fish oil in rheumatoid arthritis
`have been published (121–126), and each concluded that there is
`benefit from fish oil. In an editorial commentary discussing the
`use of fish oil in rheumatoid arthritis, it was concluded that “the
`findings of benefit from fish