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Effect of Dietary Fatty Acids on Inflammatory Gene Expression in Healthy Humans*

Open AccessPublished:April 09, 2009DOI:https://doi.org/10.1074/jbc.M109.004861
      Over the past 100 years, changes in the food supply in Western nations have resulted in alterations in dietary fatty acid consumption, leading to a dramatic increase in the ratio of omega-6 (ω6) to ω3 polyunsaturated fatty acids (PUFA) in circulation and in tissues. Increased ω6/ω3 ratios are hypothesized to increase inflammatory mediator production, leading to higher incidence of inflammatory diseases, and may impact inflammatory gene expression. To determine the effect of reducing the ω6/ω3 ratio on expression of inflammatory pathway genes in mononuclear cells, healthy humans were placed on a controlled diet for 1 week, then given fish oil and borage oil for an additional 4 weeks. Serum and neutrophil fatty acid composition and ex vivo leukotriene B4 production from stimulated neutrophils were measured at the start and end of the supplementation period and after a 2-week washout. RNA was isolated from mononuclear cells and expression of PI3K, Akt, NFκB, and inflammatory cytokines was measured by real-time PCR. A marked increase was seen in serum and neutrophil levels of long-chain ω3 PUFA concomitant with a reduction in the ω6/ω3 PUFA ratio (40%). The ex vivo capacity of stimulated neutrophils to produce leukotriene B4 was decreased by 31%. Expression of PI3Kα and PI3Kγ and the quantity of PI3Kα protein in mononuclear cells was reduced after supplementation, as was the expression of several proinflammatory cytokines. These data reveal that PUFA may exert their clinical effects via their capacity to regulate the expression of signal transduction genes and genes for proinflammatory cytokines.
      Since the beginning of the 20th century, the fatty acid composition of complex lipids (such as triglycerides) in Western diets has changed dramatically, largely due to a marked increase in the consumption of omega-6 (ω6) polyunsaturated fatty acids (PUFA)
      The abbreviations used are:
      PUFA
      polyunsaturated fatty acid
      PBMC
      peripheral blood mononuclear cell
      FAME
      fatty acid methyl ester
      LT
      leukotriene
      EPA
      eicosapentaenoic acid
      DPA
      docosapentaenoic acid
      IL
      interleukin
      TNF
      tumor necrosis factor
      AA
      arachidonic acid
      DGLA
      dihomo-γ-linolenic acid
      PI3K
      phosphatidylinositol 3-kinase.
      2The abbreviations used are:PUFA
      polyunsaturated fatty acid
      PBMC
      peripheral blood mononuclear cell
      FAME
      fatty acid methyl ester
      LT
      leukotriene
      EPA
      eicosapentaenoic acid
      DPA
      docosapentaenoic acid
      IL
      interleukin
      TNF
      tumor necrosis factor
      AA
      arachidonic acid
      DGLA
      dihomo-γ-linolenic acid
      PI3K
      phosphatidylinositol 3-kinase.
      and a concomitant decrease in the consumption of omega-3 (ω3) PUFA (
      • Kris-Etherton P.M.
      • Harris W.S.
      • Appel L.J.
      ,
      • Simopoulos A.P.
      ). The increased ingestion of ω6 PUFA is due in large part to growth in the production and consumption of vegetable oils, beef, pork, and poultry and has been exacerbated by changes in livestock husbandry and feeding practices (
      • Cordain L.
      • Watkins B.A.
      • Florant G.L.
      • Kelher M.
      • Rogers L.
      • Li Y.
      ). This together with a reduced consumption of wild, fatty fish containing high concentrations of ω3 PUFA has resulted in lower ω3 intake and in ω6/ω3 ratios in the United States diet of greater than 10:1. Anthropological evidence suggests that our hunter-gatherer ancestors maintained a ratio closer to 2:1 for ∼100,000 generations (
      • Cordain L.
      • Watkins B.A.
      • Florant G.L.
      • Kelher M.
      • Rogers L.
      • Li Y.
      ,
      • Cordain L.
      The Paleo Diet.
      ). This dietary change is postulated to enhance circulating and cellular pro-inflammatory mediators (eicosanoids and cytokines) and reduce anti-inflammatory mediators, resulting in an overall increase in systemic inflammation and a higher incidence of allergic and inflammatory disease including asthma, allergies, diabetes, cardiovascular disease, and arthritis.
      However, despite 50 years of research supporting the efficacy of long chain PUFA, such as eicosapentaenoic acid (EPA, 20:5, ω3) and docosahexaenoic acid (DHA, 20:6, ω3) in treating inflammatory diseases, a great deal remains unknown regarding the underlying molecular mechanisms responsible for their potent biological effects. The most studied mechanisms center around the observation that shifts in dietary consumption of ω6 and ω3 PUFA lead to alterations in the quantities of ω6 and ω3-derived eicosanoids produced in animals and humans, thereby disturbing the balance of lipid-based pro- and anti-inflammatory mediators produced at sites of inflammation. For example, the 4-series leukotrienes (LT) (e.g. LTB4) produced by the action of 5-lipoxygenase (5-LO) on the ω6 PUFA arachidonic acid (AA, 20:4) are highly inflammatory, while the 5-series LT (e.g. LTB5) produced from the ω3 PUFA EPA are 10–100-fold less active (
      • Miller A.M.
      • van Bekkum D.W.
      • Kobb S.M.
      • McCrohan M.B.
      • Knaan-Shanzer S.
      ). Also, Serhan et al. (
      • Serhan C.N.
      • Hong S.
      • Gronert K.
      • Colgan S.P.
      • Devchand P.R.
      • Mirick G.
      • Moussignac R.L.
      ) have demonstrated that increasing cellular uptake of ω3 fatty acids causes an enhancement in the production of resolvins and protectins, which are proposed to dampen and resolve inflammatory responses.
      Alternatively, the presence of high concentrations of ω3 PUFA, or shifts in ω6/ω3 ratios may modulate the expression of genes known to be critical to inflammatory processes. Curtis et al. (
      • Curtis C.L.
      • Hughes C.E.
      • Flannery C.R.
      • Little C.B.
      • Harwood J.L.
      • Caterson B.
      ) have shown that α-linolenic acid (LNA, 18:3, ω3), EPA, or DHA reduce the expression of genes for TNFα and IL-1β in bovine chondrocytes. Similarly, mice fed fish oil have decreased mRNA levels for numerous inflammatory cytokines including IL-1β, IL-6, and TNFα in kidney, spleen, and peritoneal macrophages (
      • Calder P.C.
      ,
      • Calder P.C.
      ). In vitro studies from our laboratory suggest that AA alters cell cycle progression and apoptosis via its capacity to regulate several members of the AP1 family of transcription factors (
      • Monjazeb A.M.
      • High K.P.
      • Connoy A.
      • Hart L.S.
      • Koumenis C.
      • Chilton F.H.
      ).
      To date, the majority of studies examining PUFA and gene expression have been carried out in isolated cell systems and a few animal studies. This raises the question of whether the observed alterations in gene expression apply to humans. The current study has utilized a dietary intervention strategy in which healthy humans were fed controlled diets including dietary supplements containing specific dosages of short chain (18 carbon) PUFA and long chain (>20 carbon) PUFA in a manner, which is consistent with our understanding of early human diets. These experiments suggest that altering circulating levels of ω6 and ω3 PUFA likely influences inflammatory responses in part by the capacity of these fatty acids or their metabolites to regulate the expression of early signal transduction genes and to block the expression of pivotal cytokines and chemokines at a transcriptional level.

      DISCUSSION

      Research over the past 50 years has shown that ω3 PUFA have potent therapeutic effects in a wide variety of inflammatory diseases, including cardiovascular disease (
      • Christensen J.H.
      • Gustenhoff P.
      • Korup E.
      • Aarøe J.
      • Toft E.
      • Møller J.
      • Rasmussen K.
      • Dyerberg J.
      • Schmidt E.B.
      ), rheumatoid arthritis (
      • Fortin P.R.
      • Lew R.A.
      • Liang M.H.
      • Wright E.A.
      • Beckett L.A.
      • Chalmers T.C.
      • Sperling R.I.
      ,
      • Kremer J.M.
      • Bigauoette J.
      • Michalek A.V.
      • Timchalk M.A.
      • Lininger L.
      • Rynes R.I.
      • Huyck C.
      • Zieminski J.
      • Bartholomew L.E.
      ), allergic disorders, and depression (
      • Su K.P.
      • Huang S.Y.
      • Chiu C.C.
      • Shen W.W.
      ). It is not clear at this time whether it is the ratio of total ω6 to ω3 PUFAs, the ratio of long chain ω6 to ω3 PUFAs, or merely the presence of high concentrations of ω3 PUFAs (long or short chain) that is most important in determining the clinical effectiveness of these dietary oils. It is also uncertain how the PUFA and their metabolites exert their effects to alter the course of inflammation in such diseases. The aim of this study was to provide both long-chain ω3 fatty acids (EPA and DHA) in fish oil and a short-chain ω6 fatty acid (GLA) in borage oil to identify the potential anti-inflammatory mechanisms by which these fatty acids exert their effects. Two recent studies suggest that EPA/GLA combinations are especially effective in critical care patients suffering from sepsis (
      • Pontes-Arruda A.
      • Aragão A.M.
      • Albuquerque J.D.
      ,
      • Singer P.
      • Theilla M.
      • Fisher H.
      • Gibstein L.
      • Grozovski E.
      • Cohen J.
      ). Specifically, addition of relatively high concentrations of these oils decreased the amount of time patients were on the ventilator, decreased the number of days patients were in the ICU, and increased overall survival.
      An important aspect of the current study was that all participants were fed a controlled diet. This diet was provided at a caloric content necessary to maintain the body weight of each study subject while keeping the fat content equal for all individuals. A previous study from our laboratory which measured changes in serum fatty acid content with age determined that a controlled diet which normalizes background ingestion of fatty acids is necessary to observe consistent changes in circulating fatty acids in relatively small numbers of people (
      • High K.P.
      • Sinclair J.
      • Easter L.H.
      • Case D.
      • Chilton F.H.
      ).
      Our studies as well as others have demonstrated that ex vivo leukotriene production by neutrophils can serve as an important pharmacodynamic marker to determine the effects of leukotriene blockers in humans (
      • Chilton F.H.
      • Patel M.
      • Fonteh A.N.
      • Hubbard W.C.
      • Triggiani M.
      ,
      • Barham J.B.
      • Edens M.B.
      • Fonteh A.N.
      • Johnson M.M.
      • Easter L.
      • Chilton F.H.
      ,
      • Johnson M.M.
      • Swan D.D.
      • Surette M.E.
      • Stegner J.
      • Chilton T.
      • Fonteh A.N.
      • Chilton F.H.
      ). Consequently leukotriene production was measured in eleven initial subjects, and we found that the fish oil/borage oil combination was sufficient to decrease leukotriene production. Moreover, this effect was reversed after the 2-week washout period (Fig. 3), similar to the reversal of ω3 and ω6 PUFA levels in serum and PMN (FIGURE 1, FIGURE 2). We next examined the influence of the supplements on the expression of enzymes directly responsible for leukotriene generation in circulating mononuclear cells, including group IV cPLA2, 5-LO, LTA4 hydrolase, and LTC4 synthase utilizing a combination of microarray technology and real-time PCR. These studies revealed no changes in the expression of any of the examined enzymes associated with leukotriene generation. This result suggests that the observed leukotriene inhibition was a consequence of the well-described effects of this type of supplementation on AA substrate availability and/or the production of alternative fatty acid substrates such as those from DGLA or EPA and not due to expression of key enzymes of leukotriene synthesis, consistent with the 2-week metabolic washout.
      Next, we evaluated whether the in vivo changes in eicosanoids could be explained by supplement-induced alterations of key signaling enzymes or transcription factors in the signal transduction pathways that lead to their biosynthesis. Surprisingly, altering PUFA ratios caused a dramatic decrease in PI3K expression and, in particular, the expression of PI3Kα and PI3Kγ. In contrast, there was no change in expression of the other PI3K isoforms, PI3Kβ or PI3Kδ, nor were there changes in message levels of the downstream effectors, Akt and NFκB. Although there was an increase in the number of lymphocytes from week 1 to week 5, this change was very small (<8%), suggesting that the changes we saw in gene expression were not due to a change in the composition of the white blood cells.
      PI3K is the initial step in a wide array of signaling pathways, controlling cellular processes from apoptosis to cell growth and differentiation, glucose transport to cell migration, and from eicosanoid and cytokine production to the leukocyte oxidative burst. Four distinct PI3K isoforms appear to be responsible for these activities. PI3Kδ and PI3Kγ are thought to play important roles in the inflammatory response (
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ,
      • Li Z.
      • Jiang H.
      • Xie W.
      • Zhang Z.
      • Smrcka A.V.
      • Wu D.
      ,
      • Sasaki T.
      • Irie-Sasaki J.
      • Jones R.G.
      • Oliveira-dos-Santos A.J.
      • Stanford W.L.
      • Bolon B.
      • Wakeham A.
      • Itie A.
      • Bouchard D.
      • Kozieradzki I.
      • Joza N.
      • Mak T.W.
      • Ohashi P.S.
      • Suzuki A.
      • Penninger J.M.
      ). The specific functions of PI3Kα and PI3Kβ are just beginning to be understood, with studies to date showing they are involved in control of glucose uptake and metabolism in muscle, liver, and adipose tissue (
      • Vivanco I.
      • Sawyers C.L.
      ).
      Only a few studies have examined the regulation of PI3K expression. PUFA such as AA have been demonstrated to affect PI3K activity and phosphorylation. Addition of AA to PC-3 prostate cancer cells has been shown to induce at least ten NFκB-regulated genes, including IL-1β, IL-6, and IL-8. Also, PI3K and Akt, both upstream regulators of the NFκB pathway, have been shown to be activated after AA addition (
      • Hughes-Fulford M.
      • Li C.F.
      • Boonyaratanakornkit J.
      • Sayyah S.
      ). To our knowledge, this is the first paper to demonstrate that altering either concentrations or ratios of circulating ω6 and ω3 PUFA directly affects the expression of specific PI3K isoforms.
      To better understand the effect of this combination of long-chain ω3 PUFA and short-chain ω6 PUFA on the immune response, message levels of several T helper 1 (Th1), Th2, and Th17 cytokines were measured from circulating mononuclear cells before and after supplementation. This area of immune regulation was examined because several studies have highlighted the connection between T cell phenotypes and PI3K expression/activation. Class IA PI3K has been shown to control the balance of the Th response by inducing the Th2 response (i.e. humoral immunity) and/or repressing the Th1 response (
      • Fukao T.
      • Tanabe M.
      • Terauchi Y.
      • Ota T.
      • Matsuda S.
      • Asano T.
      • Kadowaki T.
      • Takeuchi T.
      • Koyasu S.
      ,
      • Fukao T.
      • Yamada T.
      • Tanabe M.
      • Terauchi Y.
      • Ota T.
      • Takayama T.
      • Asano T.
      • Takeuchi T.
      • Kadowaki T.
      • Hata
      • Ji J.
      • Koyasu S.
      ). The Th1 response is thought to be supressed because PI3K inhibits IL-12 production from dendritic cells. Agonists that induce the production of IL-12 (e.g. TLR ligands such as LPS) typically activate PI3K, creating a negative feedback loop to encourage the Th2 antibody-mediated response (
      • Guha M.
      • Mackman N.
      ,
      • Herrera-Velit P.
      • Knutson K.L.
      • Reiner N.E.
      ). In addition, PI3K negatively regulates TLR signaling at the first encounter with a pathogen, thereby turning off the Th1 response that is initiated by TLRs upon endotoxin binding (
      • Moser M.
      • Murphy K.M.
      ,
      • Trinchieri G.
      ).
      The current study revealed that dietary supplementation had no effect on the Th1 cytokine IL-12 (Fig. 6A) but reduced the Th2 cytokines IL-5 and IL-10 (Fig. 6B). It has been shown that allergy is characterized by an imbalance toward the Th2 response (
      • Bach J.F.
      ); therefore, the current results suggest that altering cytokine profiles is a potential mechanism by which this fish oil/borage oil combination may influence allergic responses.
      The Th17 axis was also examined in this study. The current literature suggests Th17 immunity plays an important role in autoimmune diseases including irritable bowel disease, multiple sclerosis, and psoriasis and blocking this cytokine network protects against autoimmune disease. Fig. 6, D and E show that supplementation blocks the expression of IL-1β and IL-23, both cytokines shown to enhance Th17 differentiation. Additionally, supplementation inhibits IL-17, a product of Th17 cells (Fig. 6F). These data suggest that in healthy populations, supplementation alters the profile of cytokines known to participate in inflammatory and autoimmune responses. In combination, changes in signaling molecule expression and cytokine production may provide prolonged changes in inflammatory responses relative to the rapidly re-equilibrating levels of PUFA and their metabolites. This suggests that effects of dietary PUFA differentially impact inflammatory leukocyte functions, as LTB4 production in neutrophils was more rapidly reversed than kinase levels and cytokine production from mononuclear cells during the washout.
      It has been hypothesized that the marked shift in ω6/ω3 fatty acids in the Western diet over the past three generations may be responsible, in part, for the increase in the risk and incidence of numerous inflammatory diseases including asthma, allergic rhinitis, diabetes, and inflammatory joint diseases. Further it has been suggested that returning PUFA concentrations and ratios in human diets to those that were found during early human development or those found in less developed populations would have anti-inflammatory effects and reduce the incidence of inflammatory diseases in the population. Several mechanisms have been postulated to explain the apparent relationship between altered PUFA ratios and increased inflammation. This report demonstrates, for the first time in humans, that the expression of an early step (PI3K) in signal transduction, as well as several important downstream effectors, are significantly reduced by altering ingestion of PUFA to shift circulating ω6 to ω3 ratios. Whether this effect is due to higher overall levels of long chain ω3 PUFA or short chain ω6 PUFA or the ratio of ω6 to ω3 PUFA is beyond the scope of this in vivo study and is presently being examined in our laboratory. However, these data provide evidence that large changes in gene expression are likely an important mechanism by which PUFA exert their potent effects in clinical conditions.

      Acknowledgments

      We thank B. Undem for critical editing of the manuscript, M. Wilson for technical support with FAME analysis, and M. Pace for administrative assistance. All authors discussed the results and commented on the manuscript. K. L. W. had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. The GCRC at Wake Forest University Baptist Medical Center provided the diets and nutritional counseling to study participants and is supported by Grant Number M01-RR07122. Potential Conflicts of Interest: F. H. C. has published books Inflammation Nation, Win the War Within, and The Gene Smart Diet and is a founder and a consultant to Pilot Therapeutics Holdings, which may be partially related to his research. His conflict of interest has been disclosed to Wake Forest University Health Sciences and outside sponsors and is institutionally managed.

      REFERENCES

        • Kris-Etherton P.M.
        • Harris W.S.
        • Appel L.J.
        Circulation. 2002; 106: 2747-2757
        • Simopoulos A.P.
        J. Am. Coll. Nutr. 2002; 21: 495-505
        • Cordain L.
        • Watkins B.A.
        • Florant G.L.
        • Kelher M.
        • Rogers L.
        • Li Y.
        Eur. J. Clin. Nutr. 2002; 56: 181-191
        • Cordain L.
        The Paleo Diet.
        Wiley, Inc., New York, NY2002
        • Miller A.M.
        • van Bekkum D.W.
        • Kobb S.M.
        • McCrohan M.B.
        • Knaan-Shanzer S.
        Prostaglandins Leukot. Essent. Fatty Acids. 1993; 49: 561-568
        • Serhan C.N.
        • Hong S.
        • Gronert K.
        • Colgan S.P.
        • Devchand P.R.
        • Mirick G.
        • Moussignac R.L.
        J. Exp. Med. 2002; 196: 1025-1037
        • Curtis C.L.
        • Hughes C.E.
        • Flannery C.R.
        • Little C.B.
        • Harwood J.L.
        • Caterson B.
        J. Biol. Chem. 2000; 275: 721-724
        • Calder P.C.
        Lipids. 2001; 36: 1007-1024
        • Calder P.C.
        Proc. Nutr. Soc. 2002; 61: 345-358
        • Monjazeb A.M.
        • High K.P.
        • Connoy A.
        • Hart L.S.
        • Koumenis C.
        • Chilton F.H.
        Carcinogenesis. 2006; 27: 1950-1960
        • Chilton F.H.
        • Patel M.
        • Fonteh A.N.
        • Hubbard W.C.
        • Triggiani M.
        J. Clin. Investig. 1993; 91: 115-122
        • Metcalfe L.D.
        • Schmitz A.A.
        • Pelka J.R.
        Anal. Chem. 1966; 38: 514-515
        • Barham J.B.
        • Edens M.B.
        • Fonteh A.N.
        • Johnson M.M.
        • Easter L.
        • Chilton F.H.
        J. Nutr. 2000; 130: 1925-1931
        • Johnson M.M.
        • Swan D.D.
        • Surette M.E.
        • Stegner J.
        • Chilton T.
        • Fonteh A.N.
        • Chilton F.H.
        J. Nutr. 1997; 127: 1435-1444
        • Christensen J.H.
        • Gustenhoff P.
        • Korup E.
        • Aarøe J.
        • Toft E.
        • Møller J.
        • Rasmussen K.
        • Dyerberg J.
        • Schmidt E.B.
        BMJ. 1996; 312: 677-678
        • Fortin P.R.
        • Lew R.A.
        • Liang M.H.
        • Wright E.A.
        • Beckett L.A.
        • Chalmers T.C.
        • Sperling R.I.
        J. Clin. Epidemiol. 1995; 48: 1379-1390
        • Kremer J.M.
        • Bigauoette J.
        • Michalek A.V.
        • Timchalk M.A.
        • Lininger L.
        • Rynes R.I.
        • Huyck C.
        • Zieminski J.
        • Bartholomew L.E.
        Lancet. 1985; 1: 184-187
        • Su K.P.
        • Huang S.Y.
        • Chiu C.C.
        • Shen W.W.
        Eur. Neuropsychopharmacol. 2003; 13: 267-271
        • Pontes-Arruda A.
        • Aragão A.M.
        • Albuquerque J.D.
        Crit. Care Med. 2006; 34: 2325-2333
        • Singer P.
        • Theilla M.
        • Fisher H.
        • Gibstein L.
        • Grozovski E.
        • Cohen J.
        Crit. Care Med. 2006; 34: 1033-1038
        • High K.P.
        • Sinclair J.
        • Easter L.H.
        • Case D.
        • Chilton F.H.
        J. Nutr. Health Aging. 2003; 7: 378-384
        • Hirsch E.
        • Katanaev V.L.
        • Garlanda C.
        • Azzolino O.
        • Pirola L.
        • Silengo L.
        • Sozzani S.
        • Mantovani A.
        • Altruda F.
        • Wymann M.P.
        Science. 2000; 287: 1049-1053
        • Li Z.
        • Jiang H.
        • Xie W.
        • Zhang Z.
        • Smrcka A.V.
        • Wu D.
        Science. 2000; 287: 1046-1049
        • Sasaki T.
        • Irie-Sasaki J.
        • Jones R.G.
        • Oliveira-dos-Santos A.J.
        • Stanford W.L.
        • Bolon B.
        • Wakeham A.
        • Itie A.
        • Bouchard D.
        • Kozieradzki I.
        • Joza N.
        • Mak T.W.
        • Ohashi P.S.
        • Suzuki A.
        • Penninger J.M.
        Science. 2000; 287: 1040-1046
        • Vivanco I.
        • Sawyers C.L.
        Nat. Rev. Cancer. 2002; 2: 489-501
        • Hughes-Fulford M.
        • Li C.F.
        • Boonyaratanakornkit J.
        • Sayyah S.
        Cancer Res. 2006; 66: 1427-1433
        • Fukao T.
        • Tanabe M.
        • Terauchi Y.
        • Ota T.
        • Matsuda S.
        • Asano T.
        • Kadowaki T.
        • Takeuchi T.
        • Koyasu S.
        Nat. Immunol. 2002; 3: 875-881
        • Fukao T.
        • Yamada T.
        • Tanabe M.
        • Terauchi Y.
        • Ota T.
        • Takayama T.
        • Asano T.
        • Takeuchi T.
        • Kadowaki T.
        • Hata
        • Ji J.
        • Koyasu S.
        Nat. Immunol. 2002; 3: 295-304
        • Guha M.
        • Mackman N.
        J. Biol. Chem. 2002; 277: 32124-32132
        • Herrera-Velit P.
        • Knutson K.L.
        • Reiner N.E.
        J. Biol. Chem. 1997; 272: 16445-16452
        • Moser M.
        • Murphy K.M.
        Nat. Immunol. 2000; 1: 199-205
        • Trinchieri G.
        Annu. Rev. Immunol. 1995; 13: 251-276
        • Bach J.F.
        N. Engl. J. Med. 2002; 347: 911-920