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Arachidonic acid immunoregulation in lambs persistently infected with border disease virus
Comp. Immun. MicrobioL infect. Dis. Vol. 16, No. 4, pp. 281-287, 1993 Printed in Great Britain. All rights reserved
014%9571/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Lid
A R A C H I D O N I C ACID I M M U N O R E G U L A T I O N IN LAMBS P E R S I S T E N T L Y I N F E C T E D WITH B O R D E R DISEASE VIRUS M. M. SAWYER 1., L. L. WILLIAMS 2, A. C. ODEON 3, S. N. GIRI4 and B. I. OSBURN 1 tDepartment of Veterinary Medicine Pathology, University of California, Davis, CA 95616, U.S.A., 2Department of Pediatrics, Ohio State University College of Medicine and the Children's Hospital Research Foundation, Columbus, Ohio, U.S.A., 3Department of Animal Production, Agriculture Experiment Station, Balcare; National Institute of Agricultural Technology, 7620 Balcare, Argentina and 4Department of Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, CA 95616, U.S.A.
(Received for publication 24 March 1993) Abstract--To evaluate arachidonic acid-related immunoregulatory mechanisms during long-term persistent pestivirus infection, we measured plasma contents of leukotriene C4 (LTC4), prostaglandin D2 (PGD2) and their plasma fatty acid (FA) precursor, arachidonic acid (AA), in six lambs with congenital border disease (BD). Significantly elevated average plasma LTC4 during the first half year of life was associated with increased PDG2 when compared to uninfected control lambs. Significantly elevated total plasma esterified AA stores suggest an effective BDV-mediated prostenoid immunostimulation. However, at 1 yr old, prostenoid secretion had fallen to normal (LTC4) or below normal (PGD2) levels. In contrast, there remained significantly elevated plasma esterified AA, present as available substrate for formation of these anti-viral immunoregulatory agents. These results suggested that preventing mobilization of AA from lipid stores for effective immune responses may be a viral strategy of BD virus that is associated with long term border disease effects.
Key words: Arachidonic acid, congenital infection, congenital rubella, leukotriene, pestivirus. Rrsmnr---Six agneaux atteints d'hypomyrlogrnrse congrnitale (Border Disease) sont utilisrs afin d'rvaluer le rrle de l'acide arachidonique dans les mrchanismes immunorrgulateurs lors d'infection persistente ~i pestivirus. Pour cela, nous avons drtermin6 les concentrations plasmatiques en leucotrirne C4 (LTC4), en prostaglandine D2 (PGD2) et en leur acide gras plasmatique prrcurseur: l'acide arachidonique (AA). Au cours des six derniers mois de la vie, le taux plasmatique moyen en LTC4 est significativement plus 61ev6 chez les agneaux malades que chez les agneaux contr616s, ainsi que le taux de PGD2. L'augmentation significative de la rrserve plasmatique en AA estrrifirs suggrre une immunostimulation indirecte de BVD par l'intermrdiaire des prostrnoides. Cependant, ~i l'~lge d'un an, la srcrrtion de prostrnoides devient soit normale (LTC4) ou soit infrrieure fi la normale (PGD2). Par contre, le taux plasmatique d'AA estrrifirs reste significativement 61evr. Les AA estrrifirs sont donc disponibles comme substrat pour la synthrse des agents immunorrgulateurs anti-viraux. Ces rrsultats suggrrent que la prrvention de la mobilisation d'AA ~ partir des lipides de rrserve pour induire une rrponse immunitaire efficace peut 6tre une stratrgie employre par le virus de la BVD et qu'elle est associre aux effets ~ long terme de la border disease.
Mots-clefs: Acide arachidonique, infection congrnitale, rubrole congrnitale, leucotrirne, pestivirus. *Author for correspondence. CIMIO16~4-a
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INTRODUCTION Border disease (BD) is a condition of newborn lambs that results from congenital infection by a non-cytopathic pestivirus, border disease virus (BDV), transmitted from the dam during the first half of gestation [1]. Pestiviruses are members of the family of nonarthropod borne togaviruses which includes the human teratogen, rubella virus [2], and the most recently classified, human Hepatitis C virus [3]. All members of this viral family can cause fetal infection and may produce similar congenital anomalies in their respective animal and human hosts [4-6]. Importantly, these studies show that fetal infection with pestiviruses also causes lifetime viral persistence with an increased susceptibility to concurrent infections and early death. BDV infection manifests itself in lambs in lesions of the nervous, endocrine, immune, skeletal and integumentary systems [7]. Lambs born with BDV exhibit tonic-clonic tremors, have a hairy rather than woolly birth coat and skeletal defects as characteristic hallmarks of border disease. BDV infection of fetal tissues and glands, leading to deficiencies of the thyroid hormones, L-3,3',5-triiodothyronine (T3) and thyroxin (T4), at birth [8] and through the first half year of life [9], continue to exert an influence throughout life. A compromised cellular immune system, identified in persistently BDV-infected lambs, also renders them susceptible to concurrent diseases [7]. During chronic and persistent viral infections, the formation from activated macrophages of potent humoral immunoregulatory agents such as leukotrienes and prostaglandins is markedly increased [10-14]. This form of immunoregulation, called the arachidonic acid cascade, may be a factor in host survival during BDV persistence [10], however, delayed manifestations of congenital togavirus infection may continue to appear over time in BDV-infected lambs [7]. Arachidonic acid (AA) from plasma and circulating immune cell membrane lipoprotein stores is the 20-carbon atom fatty acid precursor of prostenoids [13]. Arachidonic acid, a long chain polyunsaturate, is complexed and stored in circulating blood lipid compartments prior to its incorporation into these immunoregulatory agents [15]. Therefore, esterified AA levels in plasma, altered in both acute and chronic infections [10, 16, 17], have a direct relationship with activation of agents from the anti-viral arachidonic acid cascade [18, 19]. We evaluated a possible contribution of the arachidonic acid cascade to host immunoregulatory strategy in lambs persistently infected with BDV. Levels of the immunoregulatory agents, prostaglandin D2 (PGD2) and ieukotriene C4 (LTC4), derived from arachidonic acid by prostaglandin synthetase and lipoxygenase respectively, and the percent of their fatty acid precursor, AA, were measured from plasma during the first year of life from persistently BDV-infected lambs and age-matched uninfected control lambs.
MATERIALS AND METHODS Animals
Six newborn cross-bred lambs, persistently infected with naturally occurring border disease virus, were maintained for 1 yr. Virus persistence was determined by co-cultivation of peripheral blood lymphocytes with Madin Darby bovine kidney cells. The cells were stained with fluorescein isothiocyanate conjugated anti-pestivirus serum (USDA-APHIS, NVSL Ames, Iowa) and a non-cytopathic pestivirus was identified. Blood was collected by jugular venipuncture. Serum was collected and frozen at -20°C. Control lambs were
Prostenoids in BDV-infectedlambs
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age-matched. Except for the 7 month control lambs which were Suffolk, all control lambs were crossbred.
Plasma total esterified fatty acid profiles Fatty acids (FA) were extracted from frozen plasma using chloroform-methanol (2:1) followed by a Folch wash, ether extraction and drying under N2. Methylation was accomplished with boron trifluoride as previously described [17]. Detection of individual methylated FA utilized a fused silica capillary column, SP2330 (Supelco, Bellfonte, Pa) and was performed on a Hewlett-Packard 5840-A gas-liquid chromatograph equipped with a dual hydrogen flame-ionization detector with an automatic integrator. The percent of each FA in the extract was calculated by the ratio of each specific FA area, determined by retention times of purchased standards (Supelco; NuChek Prep, Elysian, Minn.) to the total area of the sample. Differences between repeated samples were < 2 % .
Leukotriene C4 (L TC 4) radioimmunoassay The leukotrienes were eluted from peripheral blood leukocyte culture supernatants with ethanol, evaporated under N 2 and stored at - 20°C until RIA. LTC4 immunoreactivity was determined in a simple competitive RIA using a specific LTC4 antibody. Results obtained for the standards ere used to construct a standard (dose-response) curve. LTC4 concentrations determined by RIA were corrected for loss during extraction using their respective recoveries.
Prostaglandin De (PGD 2) radioimmunoassay PGD2 was extracted from plasma and measured by techniques reported earlier [20]. Commercial kits for measuring PGD2 by RIA were used. P G D 2 concentrations determined by RIA were corrected for loss during extraction using their respective recoveries.
Statistical analysis Statistical analyses for the PGD2, and LTC 4 values employed the two-tailed Student's t-test. Comparisons between FA profiles of BDV infected lambs and control lambs at each time period were carried out with an IBM computer statistical program using a Mann-Whitney analysis of variance with the Bonferonni correction for comparison of multiple measures. A probability value of < 0.05 was used as the level of significance. RESULTS
Plasma leukotriene C4 (LTC4) of BD V and control lambs Average leukotriene C4 (LTC4) levels of the border disease lambs (1.72 __+0.32 ng/ml) ( m e a n + S D ) at 7 months old were higher than those of the control lambs (1.05 _+ 1.57 ng/ml; P < 0.04) (Fig. 1; left panel). In contrast, LTC4 levels at 12 months old in BDV lambs (1.57 _+ 0.71 ng/ml) and control lambs (1.57 ___0.17 mg/ml) were not statistically different (Fig. 1; left panel).
Plasma prostaglandin De (PGDe) of BD V and control lambs Prostaglandin levels between BD and control lambs did not differ at 7 months (BD = 199 __+152 pg/ml; control = 155 _+ 87 pg/ml) or at 12 months (BD = 137 + 88 pg/ml; control = 180 ___87 pg/ml) (Fig. 1; right panel).
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[ ] Border Disease Lambs [ ] Controls
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Age (months) Fig. l. Plasma content of leukotriene C4 and prostaglandin D2. Leukotriene C4 levels (ng/ml) (left panel) and prostaglandin D2 (PGD2) levels (pg/ml) (right panel) or border disease lambs i (n = 6) and age-matched control lambs [] (n = 6) are compared. At 7 m o n t h s LTC4 shows a significant difference (*) between BDV and control lambs, but at 12 m o n t h s LTC4 values are similar (see Results for exact significance). The P G D 2 values are slightly higher in BDV lambs than controls at 7 m o n t h s but at 12 m o n t h s P G D 2 values of BDV lambs have fallen below normal levels.
Plasma fatty acids of BDV and control lambs Plasma fatty acids, termed FA profiles, measuring the proportions of total esterified plasma lipid stores, were carried out at 4 and 12 months old. At both samplings, similar values were found between BD-infected lambs and control lambs in proportions of the 'Table 1. Plasma fatty acids of border disease and control lambs 4 Months
12 Months
Fatty acid:~
BDVt
Control
BDVt
Control
16:0 (palmitic) 18:0 (stearic) 18:1 (oleic) 18:2 (linoleic) 20: 0
16.0 (1,2) 19.8 (1,6) 17,6" (2.1) 20.6 (2.0) 4.2* (0.7) 7.7" (1.3) 1.7" (0.4) 2.1 (0.5)
17.0 (2.3) 20.6 (1.2) 21.9 (2.3) 21.1 (2.5) 2.6 (0.5) 3.9 (0.8) 1.0 (0.2) 1.7 (0.4)
21.5 (4.5) 20.2 (3.5) 19.4 (4.9) 12.6 (3.11 3.4" (0.5) 4.4* (0.7) 1.5 (0.7) 4.7* (2.2)
26.2 (6.6) 20.9 (4.8) 18.6 (6.1) 15,4 (3.8) 1.6 (0.3) 2.4 (0.7) 1.4 (0.8) 2.0 (0,6)
2(1:4n-6 (arachidonic) 21:0 22:6n-3 (DHA)
*BDV value significantly different from control value (see Results for exact significance). tBDV = border disease virus-infected and control lamb plasma measured at 4 and 12 months of life; mean (SD) percent of total extracted plasma esterified lipoproteins. :~Fatty acids are listed by name, the number of carbons and the position of their double bonds in the carbon chain,
Prostenoids in BDV-infectedlambs
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major FA, including palmitic C 16: 0, stearic C 18 : 0, and linoleic C 18 : 2n-6 acids (Table 1). This indicated that BDV persistence had not caused disturbances in major FA metabolic pathways. However, there were consistent and significant differences in the percent of the immunoregulatory agent precursor, AA (Table 1). At both 4 and 12 months, the plasma stores of AA were significantly higher in the plasma of BD lambs than in uninfected control lambs (P < 0.002 and P < 0.004 respectively). In addition at 4 months BDV-infected lamb plasma contained a lower value of oleic C18:1, but higher values of minor fatty acids, C20:0 and C21:0 (P < 0.003), compared to control plasma (Table 1). To interpret changes in FA profiles a ratio of total unsaturated: saturated fatty acids can be calculated. At 4 months the saturation ratio of BDV lambs is 1.10 while the control lambs' ratio of 1.90 is significantly different (P < 0.01). A higher ratio indicates a higher amount of saturated FA in the plasma lipid components during early BDV persistent infection. In contrast, at 12 months in BDV plasma, oleic acid was within the normal range although C20:0 was still elevated (P < 0.004). At this time the fatty acid saturation ratios were similar between the lambs: BDV = 1.02; control = 1.14. Interestingly at 12 months, the longest unsaturated FA component of plasma (C22:6n-3; docosahexaenoic acid) was significantly higher than in control lambs (P < 0.01). DISCUSSION Although striking clinical signs lessen with age, lambs persistently infected with BDV remain severely susceptible to concurrent diseases [21]. Reduced cell-mediated immunity and lymphocyte function, shown previously [7], may be related to BDV infection of circulating immune blood cells in these lambs as indicated by the BDV infection of the peripheral blood lymphocytes persisting throughout life. We report here the additional immunoregulatory effect of an arachidonic acid cascade [10] during early (4--7 months of life) persistent BDV infection by a significantly increased amount of plasma leukotriene C4 (LTC4) and elevated plasma esterified arachidonic acid (AA) stores in BDV-infected lambs compared to age-matched control lambs. Leukotrienes formed by lipoxygenase from AA are among the major cell mediators in inflammatory reactions [13, 22]. Although precise viral mechanisms leading to host anti-viral LTC4 production are unknown, transmembrane signalling with increased binding affinity or enhanced receptor coupling may be involved [18, 23]. Viruses are also known to increase phospholipase A2 (PLA2) activity [10] an enzyme essential in cleaving AA from membrane phospholipids [15]. In addition, RNA viruses, such as Sendai and parainfluenza A2, enhance the permeability of neutrophil and macrophage membranes to calcium [12, 24], an action which is linked to PLA2 activation and arachidonic acid release [25]. We did not determine the mechanisms leading to increased LTC4 in the case of BDV-infected lambs. Increased production of prostaglandins from AA by action of prostaglandin synthase, has been also associated with chronic human viral infections [13] and in ruminants with bovine respiratory syncytial virus [26] and bluetongue virus [14]. Although the prostaglandin D2 (PGD2) level did not reach significance in our comparison of BDV and control lambs at 7 months old, the BDV level was higher than controls and we may have missed an earlier, greater PGD2 secretion. It is quite clear, however, that by 12 months, despite persistence of virus, there was no enhancement of prostenoid immunoregulatory agent
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production. L T C 4 and P G D 2 secretions in BDV lambs were similar to, or lower than, control lamb levels at 12 months. However, in contrast, it seems strange that A A stores in BD lamb plasma lipoproteins were still significantly elevated at 12 m o n t h s c o m p a r e d to controls, and presumably available for prostenoid formation. Recent understanding o f host immune system mechanisms in response to retrovirus infections m a y put these findings in perspective. Both h u m a n immunodeficiency (HIV) [27] and feline leukemia [28] viruses are capable o f blocking host phospholipid synthesis. In fact, an H I V viral strategy o f metabolic interference with production o f the precise prostenoid reagents which would eliminate them m a y lead to the development of clinical A I D S [29]. The lack o f prostenoid secretion in BDV-infected lambs despite a clear clinical need at 12 months, combined with an increased a m o u n t o f A A in readily available plasma stores, suggests a situation parallel to retrovirus behavior [27] in BD. There m a y be a block o f prostenoid production in the persistently BDV-infected lamb macrophages thus allowing B D V to evade effective host immune destruction and to maintain persistent infection. The ratio o f saturated: unsaturated F A in BDV-infected lambs at 4 m o n t h s showed an unusually high level o f saturated FA, similar to that found in another pestivirus, rubella-infected tissues [30]. However, by 12 months, the saturation ratio had returned to control levels. Since the degree o f saturation of F A o f m e m b r a n e phospholipids is vitally important to their flexibility, permeability, and function [31, 32], 12 m o n t h normalized F A profiles in B D V lambs m a y reflect homeostatic metabolic efforts o f the b o d y to stabilize m e m b r a n e lipoprotein c o m p o n e n t s to restore circulating cell m e m b r a n e functions [32, 33]. We conclude that circulating arachidonate is released to form a prostenoid immune cascade early in B D V infections. However, by the end of the first year o f life, virus-cell interactions that remain to be elucidated appear to interfere with arachidonic acid i m m u n o r e g u l a t o r y agent production despite excessive A A stores. This lack, combined with other virus-mediated immune defects, m a y explain the increased susceptibility to disease and other delayed manifestations characteristic o f B D V persistence. Acknowledgements--We are grateful for the support of the Columbus Children's Hospital Research Foundation,
the Department of Veterinary Pathobiology, The Ohio State University, Columbus, Ohio. We acknowledge the excellent technical assistance of Christy Steffen and Priscilla Powers.
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9. Sawyer M. M. and Osburn B. I. Long term effects of persistent pestivirus infection on thyroid secretions in border disease sheep. Proc. 2nd Symposium on Ruminant Pestiviruses, 1-3 October, Annecy, France (1992). 10. Plescia O. J. and Racis S. Prostaglandins as physiological immunoregulators. Prog. Allergy 44, 153-171 (1988). 1 I. Barker K., Aderem A. and Hanafusa H. Modulation of arachidonic acid metabolism by Rous sarcoma virus. J. Virol. 63, 2929-2935 (1989). 12. Laegreid W. W., Taylor S. M., Leid R. W., Silflow R. M., Evermann J. R., Breeze R. G. and Liggit H. D. Virus-induced enhancement of arachidonate metabolism by bovine alveolar macrophages in vitro. J. Leukocyte Biol. 45, 283-292 (1989). 13. Lewis R. A., Austen F. and Soberman R. J. Leukotrienes and other products of the 5-1ipogenase pathway. N. Engl. J. Med. 323, 645~55 (1990). 14. Ode6n A. C. Histamine and arachidonic acid metabolites in plasma and peripheral blood leukocyte cultures during the experimental infection of vaccine/sensitized and nonvaccinated cattle with bluetongue virus serotype ! 1. Chapter III. Ph.D. Dissertation, University of California, Davis (1991). 15. Flower R. J. and Blackswell G. J. The importance of phospholipase A2 in the prostaglandin biosynthesis. Biochem. Pharmac. 23, 285-291 (1976). 16. Kinsella J. E., Broughton K. S. and Whelan J. W. Dietary unsaturated fatty acids: interactions and possible needs in relation to eicosanoid synthesis. J. nutr. Biochem. 1, 123-141 (1990). 17. Williams L. L., Doody D. M. and Horrocks L. A. Serum fatty acid proportions are altered during the year following acute Epstein-Barr virus infection. Lipids 23, 981-988 (1988). 18. Scott W. A., Zrike J. M., Hamill A. L., Kempe J. and Cohn Z. A. Regulation of arachidonic acid metabolites in macrophages. J. exp. Med. 132, 324-335 (1980). 19. Sheaff C. M. and Holub B. J. Altered phospholipid composition of plasma cell membranes from thrombin-stimulated human platelets. Biochim. biophys. Acta 834, 164-171 (1985). 20. Giri S. L., Chen Z., Carroll E. J., Mueller R., Schiedt M. S. and Panico L. Role of prostaglandins in pathogenesis of bovine mastitis induced by Escherichia coli endotoxin. Am. J. vet. Res. 45, 586-591 (1984). 21. Gard G. P., Acland H. M. and Plant J. W. A mucosal disease virus as a cause of abortion, hairy birth coat and unthriftiness in shee~-observations on lambs surviving for longer than seven days. Aust. vet. J. 52, 64~68 (1976). 22. Morris H. R., Taylor G. W., Piper P. J. and Tippins J. R. Structure of slow-reacting substance of anaphylaxis from guinea-pig lung. Nature 285, 104-106 (1980). 23. Rola-Pleszczynski M., Gagnon L. and Chavailloz P. A. Immune regulation by leukotriene C4. Ann. N.Y. acad. Sci. 524, 218 226 (1988). 24. Dick E., Buckner C. K. and Busse W. W. The effect of parainfluenza 3 infection on guinea pig basophil and lung mast cell histamine. Am. rev. resp. Dis. 139, 715-720 (1989). 25. Samuelsson B., Goldyne M., Granstr6n E., Hamberg M., Hammarstr6n and Malmsten C. Prostaglandins and thromboxanes. Ann. rev. Biochem. 47, 997-1029 (1978). 26. Gershwin L. J., Giri S. N., Stewart R. S. and Chen J. Prostaglandin and thromboxane concentrations in plasma and lung lavage fluids during sequential infection of vaccinated and non-vaccinated calves with bovine respiratory syncytial virus. Am. J. vet. Res. 60, 1254-1262 (1989). 27. Lynn W. S., Tweedale A. and Cloyd M. W. Human immunodeficiency virus (HIV-I) cytotoxicity: perturbation of the cell membrane and depression of phospholipid synthesis. Virology 163, 43-51 (1988). 28. Rojko J. L., Fulton R. M., Rezanka L. J., Williams L. L., Copelan E., Cheney C. M., Reichel G. S., Neil J. C., Mathes L. E., Fisher T. G. and Cloyd M. W. Lymphocytotoxic strains of feline leukemia virus induce apoptosis in feline T4-thymic lymphoma cells. Lab. Invest. 66, 418-426 (1992). 29. Marcus S. G. Breakdown of PGE 1 synthesis is responsible for the acquired immunodeficiency syndrome. Med. Hypothes. 15, 39-46 (1984). 30. Voiland A. and Bardeletti G. Fatty acid composition of rubella virus and BHK21/13S infected cells. Archs Virol. 64, 319-328 (1980). 31. Spector A. A. and Yorek M. A. Membrane lipid composition and cellular function. J. lipid Res. 26, 1015-1075 (1985). 32. Yeagle P. L. Lipid regulation of cell membrane structure and function. F A S E B J. 3, 1833-1842 (1989). 33. Pagano M. E. and Sleight R. G. Defining lipid transport pathways in animal cells. Science 229, 1051-1057 (1985).
014%9571/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Lid
A R A C H I D O N I C ACID I M M U N O R E G U L A T I O N IN LAMBS P E R S I S T E N T L Y I N F E C T E D WITH B O R D E R DISEASE VIRUS M. M. SAWYER 1., L. L. WILLIAMS 2, A. C. ODEON 3, S. N. GIRI4 and B. I. OSBURN 1 tDepartment of Veterinary Medicine Pathology, University of California, Davis, CA 95616, U.S.A., 2Department of Pediatrics, Ohio State University College of Medicine and the Children's Hospital Research Foundation, Columbus, Ohio, U.S.A., 3Department of Animal Production, Agriculture Experiment Station, Balcare; National Institute of Agricultural Technology, 7620 Balcare, Argentina and 4Department of Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, CA 95616, U.S.A.
(Received for publication 24 March 1993) Abstract--To evaluate arachidonic acid-related immunoregulatory mechanisms during long-term persistent pestivirus infection, we measured plasma contents of leukotriene C4 (LTC4), prostaglandin D2 (PGD2) and their plasma fatty acid (FA) precursor, arachidonic acid (AA), in six lambs with congenital border disease (BD). Significantly elevated average plasma LTC4 during the first half year of life was associated with increased PDG2 when compared to uninfected control lambs. Significantly elevated total plasma esterified AA stores suggest an effective BDV-mediated prostenoid immunostimulation. However, at 1 yr old, prostenoid secretion had fallen to normal (LTC4) or below normal (PGD2) levels. In contrast, there remained significantly elevated plasma esterified AA, present as available substrate for formation of these anti-viral immunoregulatory agents. These results suggested that preventing mobilization of AA from lipid stores for effective immune responses may be a viral strategy of BD virus that is associated with long term border disease effects.
Key words: Arachidonic acid, congenital infection, congenital rubella, leukotriene, pestivirus. Rrsmnr---Six agneaux atteints d'hypomyrlogrnrse congrnitale (Border Disease) sont utilisrs afin d'rvaluer le rrle de l'acide arachidonique dans les mrchanismes immunorrgulateurs lors d'infection persistente ~i pestivirus. Pour cela, nous avons drtermin6 les concentrations plasmatiques en leucotrirne C4 (LTC4), en prostaglandine D2 (PGD2) et en leur acide gras plasmatique prrcurseur: l'acide arachidonique (AA). Au cours des six derniers mois de la vie, le taux plasmatique moyen en LTC4 est significativement plus 61ev6 chez les agneaux malades que chez les agneaux contr616s, ainsi que le taux de PGD2. L'augmentation significative de la rrserve plasmatique en AA estrrifirs suggrre une immunostimulation indirecte de BVD par l'intermrdiaire des prostrnoides. Cependant, ~i l'~lge d'un an, la srcrrtion de prostrnoides devient soit normale (LTC4) ou soit infrrieure fi la normale (PGD2). Par contre, le taux plasmatique d'AA estrrifirs reste significativement 61evr. Les AA estrrifirs sont donc disponibles comme substrat pour la synthrse des agents immunorrgulateurs anti-viraux. Ces rrsultats suggrrent que la prrvention de la mobilisation d'AA ~ partir des lipides de rrserve pour induire une rrponse immunitaire efficace peut 6tre une stratrgie employre par le virus de la BVD et qu'elle est associre aux effets ~ long terme de la border disease.
Mots-clefs: Acide arachidonique, infection congrnitale, rubrole congrnitale, leucotrirne, pestivirus. *Author for correspondence. CIMIO16~4-a
281
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M.M. SAWYERet
al.
INTRODUCTION Border disease (BD) is a condition of newborn lambs that results from congenital infection by a non-cytopathic pestivirus, border disease virus (BDV), transmitted from the dam during the first half of gestation [1]. Pestiviruses are members of the family of nonarthropod borne togaviruses which includes the human teratogen, rubella virus [2], and the most recently classified, human Hepatitis C virus [3]. All members of this viral family can cause fetal infection and may produce similar congenital anomalies in their respective animal and human hosts [4-6]. Importantly, these studies show that fetal infection with pestiviruses also causes lifetime viral persistence with an increased susceptibility to concurrent infections and early death. BDV infection manifests itself in lambs in lesions of the nervous, endocrine, immune, skeletal and integumentary systems [7]. Lambs born with BDV exhibit tonic-clonic tremors, have a hairy rather than woolly birth coat and skeletal defects as characteristic hallmarks of border disease. BDV infection of fetal tissues and glands, leading to deficiencies of the thyroid hormones, L-3,3',5-triiodothyronine (T3) and thyroxin (T4), at birth [8] and through the first half year of life [9], continue to exert an influence throughout life. A compromised cellular immune system, identified in persistently BDV-infected lambs, also renders them susceptible to concurrent diseases [7]. During chronic and persistent viral infections, the formation from activated macrophages of potent humoral immunoregulatory agents such as leukotrienes and prostaglandins is markedly increased [10-14]. This form of immunoregulation, called the arachidonic acid cascade, may be a factor in host survival during BDV persistence [10], however, delayed manifestations of congenital togavirus infection may continue to appear over time in BDV-infected lambs [7]. Arachidonic acid (AA) from plasma and circulating immune cell membrane lipoprotein stores is the 20-carbon atom fatty acid precursor of prostenoids [13]. Arachidonic acid, a long chain polyunsaturate, is complexed and stored in circulating blood lipid compartments prior to its incorporation into these immunoregulatory agents [15]. Therefore, esterified AA levels in plasma, altered in both acute and chronic infections [10, 16, 17], have a direct relationship with activation of agents from the anti-viral arachidonic acid cascade [18, 19]. We evaluated a possible contribution of the arachidonic acid cascade to host immunoregulatory strategy in lambs persistently infected with BDV. Levels of the immunoregulatory agents, prostaglandin D2 (PGD2) and ieukotriene C4 (LTC4), derived from arachidonic acid by prostaglandin synthetase and lipoxygenase respectively, and the percent of their fatty acid precursor, AA, were measured from plasma during the first year of life from persistently BDV-infected lambs and age-matched uninfected control lambs.
MATERIALS AND METHODS Animals
Six newborn cross-bred lambs, persistently infected with naturally occurring border disease virus, were maintained for 1 yr. Virus persistence was determined by co-cultivation of peripheral blood lymphocytes with Madin Darby bovine kidney cells. The cells were stained with fluorescein isothiocyanate conjugated anti-pestivirus serum (USDA-APHIS, NVSL Ames, Iowa) and a non-cytopathic pestivirus was identified. Blood was collected by jugular venipuncture. Serum was collected and frozen at -20°C. Control lambs were
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age-matched. Except for the 7 month control lambs which were Suffolk, all control lambs were crossbred.
Plasma total esterified fatty acid profiles Fatty acids (FA) were extracted from frozen plasma using chloroform-methanol (2:1) followed by a Folch wash, ether extraction and drying under N2. Methylation was accomplished with boron trifluoride as previously described [17]. Detection of individual methylated FA utilized a fused silica capillary column, SP2330 (Supelco, Bellfonte, Pa) and was performed on a Hewlett-Packard 5840-A gas-liquid chromatograph equipped with a dual hydrogen flame-ionization detector with an automatic integrator. The percent of each FA in the extract was calculated by the ratio of each specific FA area, determined by retention times of purchased standards (Supelco; NuChek Prep, Elysian, Minn.) to the total area of the sample. Differences between repeated samples were < 2 % .
Leukotriene C4 (L TC 4) radioimmunoassay The leukotrienes were eluted from peripheral blood leukocyte culture supernatants with ethanol, evaporated under N 2 and stored at - 20°C until RIA. LTC4 immunoreactivity was determined in a simple competitive RIA using a specific LTC4 antibody. Results obtained for the standards ere used to construct a standard (dose-response) curve. LTC4 concentrations determined by RIA were corrected for loss during extraction using their respective recoveries.
Prostaglandin De (PGD 2) radioimmunoassay PGD2 was extracted from plasma and measured by techniques reported earlier [20]. Commercial kits for measuring PGD2 by RIA were used. P G D 2 concentrations determined by RIA were corrected for loss during extraction using their respective recoveries.
Statistical analysis Statistical analyses for the PGD2, and LTC 4 values employed the two-tailed Student's t-test. Comparisons between FA profiles of BDV infected lambs and control lambs at each time period were carried out with an IBM computer statistical program using a Mann-Whitney analysis of variance with the Bonferonni correction for comparison of multiple measures. A probability value of < 0.05 was used as the level of significance. RESULTS
Plasma leukotriene C4 (LTC4) of BD V and control lambs Average leukotriene C4 (LTC4) levels of the border disease lambs (1.72 __+0.32 ng/ml) ( m e a n + S D ) at 7 months old were higher than those of the control lambs (1.05 _+ 1.57 ng/ml; P < 0.04) (Fig. 1; left panel). In contrast, LTC4 levels at 12 months old in BDV lambs (1.57 _+ 0.71 ng/ml) and control lambs (1.57 ___0.17 mg/ml) were not statistically different (Fig. 1; left panel).
Plasma prostaglandin De (PGDe) of BD V and control lambs Prostaglandin levels between BD and control lambs did not differ at 7 months (BD = 199 __+152 pg/ml; control = 155 _+ 87 pg/ml) or at 12 months (BD = 137 + 88 pg/ml; control = 180 ___87 pg/ml) (Fig. 1; right panel).
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M . M . SAWYER et al.
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[ ] Border Disease Lambs [ ] Controls
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Age (months) Fig. l. Plasma content of leukotriene C4 and prostaglandin D2. Leukotriene C4 levels (ng/ml) (left panel) and prostaglandin D2 (PGD2) levels (pg/ml) (right panel) or border disease lambs i (n = 6) and age-matched control lambs [] (n = 6) are compared. At 7 m o n t h s LTC4 shows a significant difference (*) between BDV and control lambs, but at 12 m o n t h s LTC4 values are similar (see Results for exact significance). The P G D 2 values are slightly higher in BDV lambs than controls at 7 m o n t h s but at 12 m o n t h s P G D 2 values of BDV lambs have fallen below normal levels.
Plasma fatty acids of BDV and control lambs Plasma fatty acids, termed FA profiles, measuring the proportions of total esterified plasma lipid stores, were carried out at 4 and 12 months old. At both samplings, similar values were found between BD-infected lambs and control lambs in proportions of the 'Table 1. Plasma fatty acids of border disease and control lambs 4 Months
12 Months
Fatty acid:~
BDVt
Control
BDVt
Control
16:0 (palmitic) 18:0 (stearic) 18:1 (oleic) 18:2 (linoleic) 20: 0
16.0 (1,2) 19.8 (1,6) 17,6" (2.1) 20.6 (2.0) 4.2* (0.7) 7.7" (1.3) 1.7" (0.4) 2.1 (0.5)
17.0 (2.3) 20.6 (1.2) 21.9 (2.3) 21.1 (2.5) 2.6 (0.5) 3.9 (0.8) 1.0 (0.2) 1.7 (0.4)
21.5 (4.5) 20.2 (3.5) 19.4 (4.9) 12.6 (3.11 3.4" (0.5) 4.4* (0.7) 1.5 (0.7) 4.7* (2.2)
26.2 (6.6) 20.9 (4.8) 18.6 (6.1) 15,4 (3.8) 1.6 (0.3) 2.4 (0.7) 1.4 (0.8) 2.0 (0,6)
2(1:4n-6 (arachidonic) 21:0 22:6n-3 (DHA)
*BDV value significantly different from control value (see Results for exact significance). tBDV = border disease virus-infected and control lamb plasma measured at 4 and 12 months of life; mean (SD) percent of total extracted plasma esterified lipoproteins. :~Fatty acids are listed by name, the number of carbons and the position of their double bonds in the carbon chain,
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major FA, including palmitic C 16: 0, stearic C 18 : 0, and linoleic C 18 : 2n-6 acids (Table 1). This indicated that BDV persistence had not caused disturbances in major FA metabolic pathways. However, there were consistent and significant differences in the percent of the immunoregulatory agent precursor, AA (Table 1). At both 4 and 12 months, the plasma stores of AA were significantly higher in the plasma of BD lambs than in uninfected control lambs (P < 0.002 and P < 0.004 respectively). In addition at 4 months BDV-infected lamb plasma contained a lower value of oleic C18:1, but higher values of minor fatty acids, C20:0 and C21:0 (P < 0.003), compared to control plasma (Table 1). To interpret changes in FA profiles a ratio of total unsaturated: saturated fatty acids can be calculated. At 4 months the saturation ratio of BDV lambs is 1.10 while the control lambs' ratio of 1.90 is significantly different (P < 0.01). A higher ratio indicates a higher amount of saturated FA in the plasma lipid components during early BDV persistent infection. In contrast, at 12 months in BDV plasma, oleic acid was within the normal range although C20:0 was still elevated (P < 0.004). At this time the fatty acid saturation ratios were similar between the lambs: BDV = 1.02; control = 1.14. Interestingly at 12 months, the longest unsaturated FA component of plasma (C22:6n-3; docosahexaenoic acid) was significantly higher than in control lambs (P < 0.01). DISCUSSION Although striking clinical signs lessen with age, lambs persistently infected with BDV remain severely susceptible to concurrent diseases [21]. Reduced cell-mediated immunity and lymphocyte function, shown previously [7], may be related to BDV infection of circulating immune blood cells in these lambs as indicated by the BDV infection of the peripheral blood lymphocytes persisting throughout life. We report here the additional immunoregulatory effect of an arachidonic acid cascade [10] during early (4--7 months of life) persistent BDV infection by a significantly increased amount of plasma leukotriene C4 (LTC4) and elevated plasma esterified arachidonic acid (AA) stores in BDV-infected lambs compared to age-matched control lambs. Leukotrienes formed by lipoxygenase from AA are among the major cell mediators in inflammatory reactions [13, 22]. Although precise viral mechanisms leading to host anti-viral LTC4 production are unknown, transmembrane signalling with increased binding affinity or enhanced receptor coupling may be involved [18, 23]. Viruses are also known to increase phospholipase A2 (PLA2) activity [10] an enzyme essential in cleaving AA from membrane phospholipids [15]. In addition, RNA viruses, such as Sendai and parainfluenza A2, enhance the permeability of neutrophil and macrophage membranes to calcium [12, 24], an action which is linked to PLA2 activation and arachidonic acid release [25]. We did not determine the mechanisms leading to increased LTC4 in the case of BDV-infected lambs. Increased production of prostaglandins from AA by action of prostaglandin synthase, has been also associated with chronic human viral infections [13] and in ruminants with bovine respiratory syncytial virus [26] and bluetongue virus [14]. Although the prostaglandin D2 (PGD2) level did not reach significance in our comparison of BDV and control lambs at 7 months old, the BDV level was higher than controls and we may have missed an earlier, greater PGD2 secretion. It is quite clear, however, that by 12 months, despite persistence of virus, there was no enhancement of prostenoid immunoregulatory agent
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production. L T C 4 and P G D 2 secretions in BDV lambs were similar to, or lower than, control lamb levels at 12 months. However, in contrast, it seems strange that A A stores in BD lamb plasma lipoproteins were still significantly elevated at 12 m o n t h s c o m p a r e d to controls, and presumably available for prostenoid formation. Recent understanding o f host immune system mechanisms in response to retrovirus infections m a y put these findings in perspective. Both h u m a n immunodeficiency (HIV) [27] and feline leukemia [28] viruses are capable o f blocking host phospholipid synthesis. In fact, an H I V viral strategy o f metabolic interference with production o f the precise prostenoid reagents which would eliminate them m a y lead to the development of clinical A I D S [29]. The lack o f prostenoid secretion in BDV-infected lambs despite a clear clinical need at 12 months, combined with an increased a m o u n t o f A A in readily available plasma stores, suggests a situation parallel to retrovirus behavior [27] in BD. There m a y be a block o f prostenoid production in the persistently BDV-infected lamb macrophages thus allowing B D V to evade effective host immune destruction and to maintain persistent infection. The ratio o f saturated: unsaturated F A in BDV-infected lambs at 4 m o n t h s showed an unusually high level o f saturated FA, similar to that found in another pestivirus, rubella-infected tissues [30]. However, by 12 months, the saturation ratio had returned to control levels. Since the degree o f saturation of F A o f m e m b r a n e phospholipids is vitally important to their flexibility, permeability, and function [31, 32], 12 m o n t h normalized F A profiles in B D V lambs m a y reflect homeostatic metabolic efforts o f the b o d y to stabilize m e m b r a n e lipoprotein c o m p o n e n t s to restore circulating cell m e m b r a n e functions [32, 33]. We conclude that circulating arachidonate is released to form a prostenoid immune cascade early in B D V infections. However, by the end of the first year o f life, virus-cell interactions that remain to be elucidated appear to interfere with arachidonic acid i m m u n o r e g u l a t o r y agent production despite excessive A A stores. This lack, combined with other virus-mediated immune defects, m a y explain the increased susceptibility to disease and other delayed manifestations characteristic o f B D V persistence. Acknowledgements--We are grateful for the support of the Columbus Children's Hospital Research Foundation,
the Department of Veterinary Pathobiology, The Ohio State University, Columbus, Ohio. We acknowledge the excellent technical assistance of Christy Steffen and Priscilla Powers.
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