Animal Reproduction Science 82–83 (2004) 281–294 Steroidal regulation of uterine immune defenses G.S. Lewis∗ United States Department of Agriculture, Agricultural Research Service, U.S. Sheep Experiment Station, HC 62 Box 2010, Dubois, ID 83423, USAAbstract Progesterone suppresses uterine immune defenses and predisposes postpartum animals to non-speciﬁc uterine infections. Progesterone can also suppress uterine eicosanoid synthesis. This ef-fect of progesterone seems to be an important factor in the onset of uterine infections becauseeicosanoids can enhance uterine immune defenses. In fact, exogenous prostaglandin F2 (PGF2 ),an eicosanoid that stimulates uterine PGF2 production, enhances uterine immune defenses andpromotes the ability of ewes and sows to resolve uterine infections, even when progesterone ismaintained at luteal phase concentrations. Prostaglandin F2 is also a proinﬂammatory moleculethat stimulates the production of proinﬂammatory cytokines and may enhance uterine production ofleukotriene B4 (LTB4 ), which stimulates various neutrophil functions. Neutrophils seem to mountthe initial response to bacteria that enter the uterus, and proinﬂammatory cytokines and LTB4 en-hance phagocytic activity of neutrophils. Even though there are clear associations among PGF2 ,LTB4 , proinﬂammatory cytokines, phagocytosis, and the ability of the uterus to resist or resolveinfections, the mechanisms of action of exogenous PGF2 in mitigating the immunosuppressiveeffects of progesterone have not yet been deﬁned. However, deﬁning the PGF2 mechanisms shouldyield important new information that can be used to develop novel prevention and treatment strate-gies that do not rely on antibiotic and antimicrobial compounds for managing uterine infections.Published by Elsevier B.V.Keywords: Uterus; Infections; Progesterone; Prostaglandins; Eicosanoids1. Introduction Progesterone suppresses uterine immune defenses and predisposes the uterus to nonspe-ciﬁc infections. This occurs most commonly in postpartum animals, and postpartum uterineinfections may reduce the reproductive performance of livestock (Arthur et al., 1989; Lewis, ∗ Tel.: +1-208-374-5306; fax: +1-208-374-5582.E-mail address: email@example.com (G.S. Lewis).0378-4320/$ – see front matter. Published by Elsevier B.V.doi:10.1016/j.anireprosci.2004.04.026
282 G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–2941997; Dhaliwal et al., 2001). Even though the immunosuppressive effects of progesteronehave been recognized for at least 50 years (Black et al., 1953; Rowson et al., 1953), thecomplete mechanisms of action of progesterone are not known. However, recent researchhas revealed important clues about how progesterone suppresses uterine immune defenses,and those clues are critical for developing new prevention and treatment strategies that donot rely on antibiotic and antimicrobial compounds for managing uterine infections. Uterine infections are called nonspeciﬁc when numerous potentially pathogenic bacteriacan be isolated from infected uteri; the initial colonizing bacteria are not known; and thespeciﬁc bacteria that cause the signs of infection are not known (Grifﬁn et al., 1974a,b; DelVecchio et al., 1994; Lewis, 1997). Despite that, Arcanobacterium pyogenes and Escherichiacoli are most often associated with spontaneous uterine infections in livestock (Grifﬁn et al.,1974a,b; Del Vecchio et al., 1994; Dhaliwal et al., 2001). Thus, A. pyogenes and E. coli areoften used to induce uterine infections for studies designed to deﬁne the permissive role of,for example, progesterone (Lewis, 2003; Wulster-Radcliffe et al., 2003). The annual incidences of uterine infections in postpartum animals range from 10 to 50% ofthe dairy cattle (Arthur et al., 1989; Lewis, 1997), 20 to 75% of the dairy buffaloes (Usmaniet al., 2001), and 5 to 10% of the dairy sheep (Tzora et al., 2002) in a given herd or ﬂock. Pub-lished incidences of similar uterine infections, rather than the mastitis–metritis–agalacticasyndrome, in postpartum sows are not available. However, based on personal communi-cations with swine herd managers and veterinarians and scientists with swine geneticscompanies, uterine infections seem to be “common,” but the speciﬁcs are often consideredto be proprietary. Nevertheless, one study indicates that uterine infections during the lutealphase in pigs are associated with increased embryonal deaths (Scoﬁeld et al., 1974), andanother report indicated that 42% of the gilts and 39% of the sows in Finland were culledbecause of impaired fertility, which was the most common reason for culling female pigs(Heinonen et al., 1998). Even though numerous authors have reported the incidences of post-partum uterine infections for cattle, buffaloes, and sheep, the reports are rough estimates.The “true” incidence of uterine infections for any livestock species is not known. This isbecause detection and diagnosis are often inaccurate; most postpartum animals are not eval-uated for signs of uterine infections; and uterine infections are not considered contagious,as is brucellosis, for example, so reporting is not mandatory (Lewis, 1997). Intramuscular (i.m.) injections of prostaglandin F2 (PGF2 ) are an efﬁcacious treatmentfor pyometra in cattle (Lewis, 1997; Sheldon and Noakes, 1998). Pyometra seems to be themost common type of uterine infection in dairy cattle, and pyometra is the type typicallyassociated with impaired reproductive performance (Lewis, 1997). Pyometra is deﬁnedsimply as pus in the uterus, but, without extensive clinical and histological evaluations,one cannot determine whether all layers of the uterine wall are involved and whether thepathogens that caused the pus to form in the uterus have escaped into the body cavity andthe circulatory system. Thus, the term can be applied to a condition with a wide range ofconsequences. Progesterone plays a permissive role in the onset of pyometra, which usuallydevelops coincidently with luteal function during the postpartum period (Lewis, 1997). Thebelief is that potentially pathogenic bacteria that reside in livestock environments enter theuterus during or after calving. Cows with assisted births and cows in which retained fetalmembranes are removed manually seem to be the most vulnerable because attendants, ineffect, inoculate the uterus with bacteria (Lewis, 1997). In cows that develop pyometra, the
G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 283introduced bacteria apparently reside in the uterus, without proliferating into an infection,until luteal progesterone suppresses uterine immune functions. The bacteria are then ableto proliferate and produce the signs of infection. Pyometra usually persists until luteolysis,when reduced progesterone concentrations no longer suppress uterine immune defensesand the uterus is able to resolve the infection. Because it is luteolytic, exogenous PGF2is injected to reduce progesterone concentrations and promote the resolution of uterineinfections. However, the true mechanism of action of PGF2 in resolving uterine infectionsis not known. In fact, PGF2 has effects that are not related to its effects on luteal function. Despite the fact that exogenous PGF2 is efﬁcacious, intrauterine and systemic antibiotictreatments are still common, and new antibiotic treatments are being introduced (Lewis,1997; Sheldon and Noakes, 1998; Chenault et al., 2001). Genuine concerns about antibioticuse in livestock and the potential for creating antibiotic-resistant strains of bacteria havefocused our research on determining whether nonantibiotic, native compounds will enhancehost immunity and prevent or resolve uterine infections. Because of the role of progesteronein making the uterus susceptible to infections, determining its mechanisms of action isessential for formulating methods to enhance the ability of the uterus to control pathogenicbacteria. Therefore, this article is a brief review of the role of progesterone in convertingthe uterus from an organ that is resistant to one that is susceptible to infections and of howeicosanoids may be used to mitigate the immunosuppressive effects of progesterone.2. Role of progesterone The uterus in cattle, sheep, and pigs is susceptible to infections when progesterone con-centrations are increased, and it is resistant to infections when progesterone concentrationsare decreased. Numerous authors have reported this during the last 50 years (Black et al.,1953; Rowson et al., 1953; Hawk et al., 1961, 1964; Lander Chacin et al., 1990; Sealset al., 2002a; Wulster-Radcliffe et al., 2003). Spontaneous uterine infections in dairy cows,for example, are not typically detected until after the ﬁrst postpartum corpus luteum formsand begins producing progesterone (Lewis, 1997). However, the uterine bacterial load canbe great enough to cause puerperal metritis shortly after calving, before progesterone con-centrations increase (Arthur et al., 1989; Lewis, 1997; Seals et al., 2002a). In postpartumbeef cows, intrauterine infusions of A. pyogenes and E. coli did not produce infections,unless progesterone concentrations had started to increase (Del Vecchio et al., 1992). Afterprogesterone concentrations had begun to increase, all of the cows developed infectionsafter intrauterine infusions of A. pyogenes and E. coli (Del Vecchio et al., 1992). Intrauter-ine infusions of A. pyogenes and E. coli into postpartum ewes did not produce infectionswhen progesterone concentrations were basal (Seals et al., 2002b; Lewis, 2003). However,infections developed in all postpartum ewes that had spontaneous luteal function and allpostpartum ewes that had been ovariectomized and treated i.m. with progesterone beforeintrauterine infusions of A. pyogenes and E. coli (Seals et al., 2002b; Lewis, 2003). Further-more, ewes and gilts that received intrauterine infusions of A. pyogenes and E. coli duringestrus did not develop uterine infections, whereas all ewes and gilts receiving intrauterineinfusions of A. pyogenes and E. coli during the luteal phase developed infections (Ramadanet al., 1997; Seals et al., 2003; Wulster-Radcliffe et al., 2003).
284 G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 Neutrophils seem to mount the initial response to bacteria that enter the uterus, and theability of neutrophils to respond to intrauterine bacteria may be the most critical componentof the uterine immune defense mechanism (Hussain, 1989; Saad et al., 1989; Hussain andDaniel, 1991). Despite that, lymphocyte proliferation in vitro is commonly used as a generalmeasure of uterine immune function (Hussain, 1989; Saad et al., 1989; Hussain and Daniel,1991; Slama et al., 1991; Cai et al., 1994). Unstimulated, concanavalin A (Con A)-stimulated(stimulates T-cells), and lipopolysaccharide (LPS)-stimulated (stimulates B-cells) lympho-cyte proliferation were all greater for cells collected from vena caval blood from postpartumewes that were ovariectomized before luteal function was detected than they were for cellscollected from vena caval blood from ovary-intact postpartum ewes (Lewis, 2003). (Unlessstated otherwise, the number of lymphocytes added to each culture well was ﬁxed in eachstudy cited. For the purposes of this article, words such as greater, increased, decreased,etc. refer to comparisons with P-values of less than 0.05.) We use procedures describedin Benoit and Dailey (1991) to collect vena caval blood through catheters that are posi-tioned just cranial to the entry of uteroovarian blood; blood from this site is enriched withuteroovarian blood. Moreover, exogenous progesterone, compared with sesame oil treat-ment, reduced unstimulated and Con-A stimulated lymphocyte proliferation in postpartumewes (Lewis, 2003). In addition, unstimulated, Con A-stimulated, and LPS-stimulated lym-phocyte proliferation were greater when cells were collected from ewes during estrus thanwhen they were collected during the luteal phase, and unstimulated and Con A-stimulatedlymphocyte proliferation were greater when cells were collected from gilts during estrusthan when cells were collected from gilts during the luteal phase (Ramadan et al., 1997;Wulster-Radcliffe et al., 2003). Other authors have reported similar data (Segerson andGunsett, 1993; Hansen, 1998; Szekeres-Bartho et al., 2001; Par et al., 2003). These effectsof exogenous and endogenous progesterone on lymphocyte proliferation were associatedwith the inability of the uterus to prevent the development of infections (Ramadan et al.,1997; Seals et al., 2002b, 2003; Lewis, 2003; Wulster-Radcliffe et al., 2003). Progesterone clearly changes the uterus from an organ that is resistant to one that issusceptible to infections. The literature cited above can be used to support the hypothesisthat the uterus “defaults” to “resistant to infections” when progesterone concentrationsare basal and bacterial contamination is not severe enough to overwhelm uterine immunedefenses, as it seems to do with puerperal metritis. We call this a “protected period,” eventhough we recognize that the protection is not absolute and some unknown load of bacteria islikely to overwhelm uterine immune defenses during this period. We have used postpartumand seasonally anestrous ewes to test hypotheses about the so called protected period. To test hypotheses about the so called protected period, autumn-lambing ewes wereovariectomized on day 9 or day 14 postpartum, which was before detection of spontaneousluteal function and increased progesterone concentrations, treated i.m. with canola oil orsafﬂower oil, and given intrauterine infusions of A. pyogenes and E. coli (Seals et al., 2002b;Lewis, 2003). None of these ewes developed uterine infections (Seals et al., 2002b; Lewis,2003). However, all of the ewes in the same experiments with spontaneous luteal functionand all of the ewes given exogenous progesterone i.m. in canola oil or safﬂower oil devel-oped infections in response to intrauterine A. pyogenes and E. coli infusions (Seals et al.,2002b; Lewis, 2003). Concentrations of PGF2 in vena caval blood collected from the ewesovariectomized on day 14 were inversely related to progesterone concentrations: PGF2
G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 285was greatest in ewes with the least vena caval progesterone; and lowest in ewes with thegreatest progesterone (Lewis, 2003). The inverse relationship between PGF2 and proges-terone is consistent with results from a study in which 6 -methyl-17 -hydroxyprogesteroneacetate, which is commonly used to control the estrous cycle, reduced uterine PGF2 pro-duction (Fort´n et al., 1994). For the day 14 postpartum ewes, ovariectomy increased Con A- ıand LPS-stimulated lymphocyte proliferation, but exogenous progesterone decreased ConA-stimulated proliferation (Lewis, 2003). Even though exogenous progesterone suppressedlymphocyte proliferation somewhat for ewes ovariectomized on day 9, the results were notas clear as they were for day-14 postpartum ewes (Seals et al., 2002b). Overall, susceptibil-ity to uterine infections was associated with increased progesterone concentrations, reducedPGF2 production, and reduced lymphocyte proliferation in vitro. By contrast, resistance touterine infections was associated with basal progesterone concentrations, increased PGF2production, and increased lymphocyte proliferation in vitro. Thus, the “default” uterineimmune defenses were adequate to prevent infections, and the idea of a protected period isnot inappropriate. We have also used seasonally anestrous ewes to determine whether the uterus is “protected”from infections in the long-term absence of ovarian progesterone and estradiol (Mink et al.,2003). These ewes had not been detected in estrus for at least three months, had basalendogenous progesterone concentrations, and had no ovarian follicles large enough to beestrogen active. The ewes were treated with either progesterone in safﬂower oil or safﬂoweroil alone and given intrauterine infusions of A. pyogenes and E. coli. Control ewes did notdevelop infections after intrauterine infusion of A. pyogenes and E. coli, but all ewes treatedwith progesterone developed uterine infections after intrauterine A. pyogenes and E. coliinfusions (Mink et al., 2003). The A. pyogenes and E. coli were infused 2 days before pro-gesterone injections were initiated to determine whether the bacteria would be eliminatedshortly after they were introduced or whether the bacteria could reside in the uterus withoutproliferating into an infection. Enough of the bacteria were clearly able to survive in theuterus until exogenous progesterone suppressed the uterine immune defenses and permittedthem to proliferate into uterine infections. Data from our studies with postpartum and seasonally anestrous ewes, in which proges-terone concentrations were basal, seem to support the idea that the uterus “defaults” to beingresistant to A. pyogenes and E. coli infections, unless a compound, such as progesterone,actively suppresses uterine immune defenses. Even though default uterine immune defensesseemed sufﬁcient to prevent frank A. pyogenes and E. coli infections, they did not eliminatethe bacteria from the uterus during the 2-day interval from bacteria infusion to initiationof progesterone treatments. The ability of A. pyogenes and E. coli to remain “quiescent”in the uterus until progesterone suppresses the uterine immune defenses seems consistentwith the scenario that has been described for postpartum dairy cows that develop uterineinfections coincident with the onset of luteal function (Lewis, 1997). Thus, the seasonallyanestrous ewe model would seem to offer the opportunity to test a number of hypothesesabout the onset of uterine infections in livestock. The temporal relationships between reductions in progesterone, increases in estradiol, orphase of the estrous cycle before hormone assays became routine, and resistance to uterineinfections have been recognized for more than 40 years (Hawk et al., 1961, 1964; Brinsﬁeldet al., 1964, 1967; Ramadan et al., 1997; Wulster-Radcliffe et al., 2003). However, a direct
286 G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294effect of estradiol on the resistance or susceptibility of the uterus to infections has not beenclearly established for cattle, sheep, or pigs. In fact, in two recent studies, estradiol didnot affect uterine involution in postpartum cattle and sheep, and the authors speculatedthat estradiol treatment would not improve uterine health (Sheldon et al., 2003a,b). Thus,because progesterone unequivocally suppresses uterine immune defenses and the role ofestradiol has not been established, progesterone seems to be the primary ovarian steroidgoverning the susceptibility of the uterus to pathogenic bacteria.3. Role of eicosanoids Prostaglandin F2 and its various analogues have been used to resolve uterine infections inlivestock, but, as already stated, its true mechanism of action is not known. Other eicosanoidshave also been evaluated, and some may promote uterine health. Eicosanoids, which includeprostaglandins and leukotrienes, are members of a large family of compounds that aresynthesized from arachidonic acid through the cyclooxygenase and lipoxygenase pathways(Pace-Asciak and Granström, 1983; Müller-Peddinghaus and Kast, 1996). Because thefamily is so large, only eicosanoids that seem to have an obvious role in regulating uterineimmune defenses will be discussed. Relationships among jugular progesterone, jugular 13,14-dihydro-15-keto-PGF2(PGFM), which is a metabolite of PGF2 , and onset of uterine infections have been charac-terized in postpartum dairy cows (Del Vecchio et al., 1992, 1994; Nakao et al., 1997; Sealset al., 2002a). The half-life of PGFM is approximately 15 min, compared with approxi-mately 1 min for PGF2 , and jugular PGFM concentrations closely reﬂect uterine PGF2production during the postpartum period, but not during the estrous cycle when the uterusproduces considerably less PGF2 (Williams et al., 1983; Guilbault et al., 1984; Fort´n et al., ı1994; Wade and Lewis, 1996). Jugular PGFM concentrations were reduced in postpartumdairy cows that subsequently developed uterine infections, compared with PGFM concen-trations in cows that did not develop uterine infections (Nakao et al., 1997; Seals et al.,2002a). Small increases in progesterone, probably luteal, preceded the onset of uterine in-fections (Seals et al., 2002a), and PGFM concentrations increased at the onset of uterineinfections (Del Vecchio et al., 1992; Seals et al., 2002a). Increased PGFM concentrationswere probably due to uterine inﬂammation in response to the growth of bacteria and releaseof endotoxin (Roitt et al., 1998; Leung et al., 2001). The studies with dairy cows and sheep seem to indicate that progesterone–PGF2 in-teractions, and not just progesterone, are important for the regulation of uterine immunedefenses and the ability of the uterus to prevent infections. Indeed, uterine PGF2 pro-duction seems to be related to the ability of the uterus to prevent or resolve infections.One hypothesis that combines those ideas is that progesterone suppresses uterine immunedefenses and prevents the uterus from resisting infections, but PGF2 , and most likelyother eicosanoids such as leukotriene B4 (LTB4 ), can enhance uterine immune defensesand mitigate the effects of progesterone. In vitro experiments lend support to that hypoth-esis; PGF2 , LTB4 , 5-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, andlipoxin B4 are chemoattractant to neutrophils (Hoedemaker et al., 1992). Indeed, neutrophilsseem to mount the initial defense against intrauterine pathogens, and suppressed neutrophil
G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 287functions seem to make the uterus susceptible to infections (Hussain, 1989; Hussain andDaniel, 1991; Cai et al., 1994). Another eicosanoid, PGE2 , can suppress immune func-tions, including neutrophil functions, and predispose cows to uterine infections (Slamaet al., 1991). Progesterone–eicosanoid interactions are clearly broad and are likely to in-volve a variety of compounds in addition to PGF2 . Thus, because numerous compoundscan interact with progesterone and affect immune functions, our research has focused onprogesterone–PGF2 interactions. As is widely known, exogenous PGF2 induces luteolysis, which reduces circulatingprogesterone, and permits the uterus to clear infections. However, exogenous PGF2 hashelped resolve uterine infections in cows without luteal function (Del Vecchio et al., 1994).Clinical veterinarians often speculate that PGF2 injections stimulate uterine contractionsthat expel bacteria from the uterus (personal communications). But this idea ignores thefacts that uterine contractions per se are not likely to kill the bacteria causing the infection;bacteria are likely to remain in the uterus and proliferate again with the next bout of lutealfunction; and published literature does not support the idea that uterine contractions cleansethe uterus. Indeed, a recent study with cows indicates that a PGF2 analogue, cloprostenol,enhances uterine contractions for perhaps 45 min after injection, and intrauterine pressurewas increased for only 15 min after injection (Hirsbrunner et al., 2003). Moreover, a studyof uterine infections in mares indicates that uterine contractions may reduce the volumeof ﬂuid in the uterus, but they do not eliminate the bacteria (Nikolakopoulos and Watson,1999). However, based on a good deal of literature, one may speculate that exogenous PGF2has direct effects on uterine immune defenses, which can eliminate bacteria. Nevertheless,studies are needed to separate the effects of uterine contractions from the direct effects ofPGF2 on complete resolution of uterine infections. A critical issue that is often overlooked in discussions about the role of exogenous PGF2in uterine infections is the fact that the direct effects of exogenous PGF2 on uterine immunedefenses and the effects of PGF2 on luteal function and progesterone concentrations arecompletely confounded. We have conducted studies with sheep and pigs to separate theeffects of exogenous PGF2 on luteal function and uterine immune defenses and address thequestions: Is exogenous PGF2 effective because it enhances uterine immune defenses andmitigates the effects of progesterone, or is exogenous PGF2 effective because it decreasesprogesterone concentrations? We have used pigs and sheep to answer those questions. We selected pigs because PGF2is not luteolytic in pigs until after approximately day 12 of the estrous cycle (Guthrie andPolge, 1976). Sows were assigned to a 2×2 factorial array of treatments (n = 6 sows/group)to determine whether PGF2 had direct effects on uterine immune defenses (unpublishedresearch). The two main effects were intrauterine infusion of A. pyogenes and E. coli (i.e.,bacteria versus phosphate-buffered saline (PBS)) and PGF2 (i.e., 10 mg of PGF2 versussaline). Bacteria or PBS was infused on day 7 of the estrous cycle, and PGF2 or salinewas injected i.m. on day 9 of the same cycle. Uteri were collected on day 11 of the cy-cle, two days after PGF2 or saline injections. Progesterone concentrations in vena cavalblood did not differ among groups and averaged 64 ng/mL during the study, indicating thatPGF2 did not affect luteal function. Vena caval estradiol-17 concentrations did not dif-fer among groups and averaged 1 ng/mL. Injection of PGF2 increased vena caval PGF2concentrations, which is consistent with data for sheep after exogenous PGF2 (Wade and
288 G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294Lewis, 1996). Unstimulated and LPS-stimulated lymphocyte proliferation also increasedin sows that received exogenous PGF2 , even though exogenous PGF2 increased venacaval PGE2 concentrations. The bacteria-treated sows developed uterine infections, but thePBS-treated sows did not develop uterine infections. Based on criteria used to determinewhether an experimental animal has a uterine infection (i.e., volume of sediment, whichcontains leucocytes, bacteria, and cellular debris from the uterus, in uterine ﬂushings col-lected postmortem and the ability to culture of A. pyogenes and E. coli from the ﬂushings),PGF2 -treated sows were resolving the uterine infections at the time of slaughter (sedi-ment volume, as a percentage of total ﬂushing volume: 70% for bacteria-saline, 30% forbacteria-PGF2 , and <5% for PBS-saline and PBS-PGF2 sows). These results indicate thatexogenous PGF2 enhanced uterine immune defenses and allowed the uterus to begin re-solving the infections, despite luteal phase progesterone concentrations and basal estradiolconcentrations. In another study to test the hypothesis that PGF2 has effects on uterine immune defensesthat are independent of progesterone concentrations, we used ovariectomized, progesterone-treated ewes (unpublished research). This model was necessary because PGF2 is luteolyticin sheep after approximately day 4 of the estrous cycle. The treatment groups were in a 2×2×2 factorial array (n = 8 ewes/group). The main effects were ovariectomy (i.e., ovariectomyversus sham procedure), progesterone (5 mg of progesterone at 12-h intervals versus sesameoil diluent at the same times), and PGF2 (15 mg of PGF2 versus saline). Ewes were eitherovariectomized or a sham procedure was performed on day 0 of the estrous cycle (i.e., dayof estrous onset). Progesterone or sesame oil was injected i.m. from day 0 through day 11.A. pyogenes and E. coli were infused intrauterine on day 6, and PGF2 or saline was injectedi.m. on day 9. Uteri were collected on day 12. All days are relative to day 0. Progesteroneconcentrations in vena caval blood were as anticipated. Progesterone concentrations in shamovariectomy–sesame oil–saline ewes were typical for luteal phase sheep. Exogenous PGF2induced luteolysis and reduced progesterone concentrations in ewes that did not receiveexogenous progesterone. Ovariectomy reduced progesterone to basal concentrations, andexogenous progesterone maintained or increased progesterone concentrations. ExogenousPGF2 increased vena caval PGF2 concentrations. All sham–ovariectomy ewes developeduterine infections, but sham–oil–saline ewes and sham–oil–PGF2 ewes were resolving theinfections by day 12 (sediment volume approximately 8%). The sham–progesterone–salineewes had severe uterine infections on day 12 (sediment volume of 28%, which is muchgreater than usual for sheep with uterine infections), but sham–progesterone–PGF2 ewesseemed to be resolving their infections on day 12 (sediment volume of approximately15%). The ovariectomy–oil–saline ewes did not have uterine infections on day 12 (sedi-ment volume of 5%), but the ovariectomy–progesterone–saline ewes had typical infections(sediment volume of approximately 16%) on day 12. The ovariectomy–oil–PGF2 ewesdid not have uterine infections on day 12 (sediment volume of approximately 2%), andthe ovariectomy–progesterone–PGF2 ewes had nearly resolved the infections (sedimentvolume of approximately 4%) by day 12. Progesterone reduced unstimulated lymphocyteproliferation, and PGF2 increased unstimulated, Con A-stimulated, and LPS-stimulatedlymphocyte proliferation. Based on the data, we reasoned that exogenous PGF2 enhancedthe ability of the uterus to resolve infections, regardless of progesterone concentrations.The sows and ewes in the two experiments selected as examples received only one PGF2
G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 289injection, and this seemed to be enough to initiate the resolution of the uterine infections.A second injection of PGF2 , 12–24 h after the ﬁrst, might have resolved all of the inducedinfections, but we have not yet conducted experiments to test that possibility. Data from recent studies support the idea that exogenous PGF2 has direct effects onuterine immune defenses: effects that are independent of luteal function and progesteroneconcentrations. In addition, the ability of the uterus to secrete PGF2 , and perhaps othereicosanoids, may govern the ability of the uterine immune defenses to resist or resolveuterine infections. Thus, it seems likely that PGF2 can enhance uterine immune defensesand mitigate the immunosuppressive effects of progesterone.4. Eicosanoid–progesterone relationships Eicosanoids and progesterone seem to have many independent effects on the activityof immune cells. Progesterone is typically immunosuppressive, and that phenomenon hasbeen studied and reviewed extensively. Progesterone regulation of the synthesis of immuno-suppressants and blocking factors has received particular attention, so those data will notbe reviewed in this article (Segerson and Gunsett, 1993; Hansen, 1998; Szekeres-Barthoet al., 2001). Rather, this section of this article will contain information about eicosanoid–progesterone relationships. Indeed, understanding these relationships at the cellular ormolecular level and using the information to develop methods to mitigate the immuno-suppressive effects of progesterone has considerable potential for preventing or resolvinguterine infections in livestock environments. The uterus is normally able to prevent potentially pathogenic bacteria introduced dur-ing estrus from proliferating into infections (Ramadan et al., 1997; Wulster-Radcliffe et al.,2003). During estrus, when progesterone concentrations are decreased and estradiol concen-trations are increased, uterine PGF2 and endometrial leukotriene production are increased(Ottobre et al., 1980; Kindahl et al., 1984; Zarco et al., 1988; Vagnoni et al., 2001). As lutealfunction develops and progesterone concentrations begin to increase, uterine PGF2 andLTB4 production decrease to basal levels within a few days after estrus, and the uterus be-comes susceptible to infections (Kindahl et al., 1984; Zarco et al., 1988; Slama et al., 1993;Vagnoni et al., 2001). In vitro studies indicate that PGF2 enhances neutrophil chemotaxisand the ability of neutrophils to ingest bacteria, and LTB4 enhances chemotaxis, randommigration, and antibody-independent cell-mediated cytotoxicity (Hoedemaker et al., 1992).These effects alone, assuming they occur in vivo, on neutrophil functions should help theuterus manage pathogens. But, in addition to direct effects on neutrophils, PGF2 is aproinﬂammatory molecule that may stimulate production of proinﬂammatory cytokinesthat enhance phagocytosis and lymphocyte functions (Kelly et al., 2001; Seals et al., 2003).Furthermore, a study with cows indicates that LTB4 promotes uterine involution and reducesthe risk of uterine infections (Slama et al., 1993). Prostaglandin F2 injections increase uterine PGF2 and luteal LTB4 production(Steadman and Murdoch, 1988; Wade and Lewis, 1996). Even though deﬁnitive data are notavailable, one may speculate that PGF2 could enhance uterine LTB4 production becausenordihydroguaiaretic acid, which inhibits lipoxygenase activity and LTB4 production, pro-longed the luteal phase in cattle and sheep and the uterus seems to have been the mediator
290 G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294(Milvae et al., 1986; Cooke and Ahmad, 1998). Injections of PGF2 that increase uterinePGF2 production probably increase uterine phospholipase A2 (PLA2 ) and cyclooxygenase2 activities, which would produce free arachidonic acid and then convert the arachidonicacid to PGF2 (Binelli et al., 2000; Diaz et al., 2002; Narayansingh et al., 2002). The freearachidonic acid could also be used to produce additional cyclooxygenase products andlipoxygenase (e.g., LTB4 ) products. In addition, tumor-necrosis factor (TNF ) mediatesinﬂammatory and cytotoxic responses (Roitt et al., 1998) and stimulates endometrial PGF2production; PLA2 seems to be the mediator (Miyamoto et al., 2000; Skarzynski et al., 2000). Thus, based on a considerable amount of literature, methods for promoting uterine PGF2and LTB4 production should enhance immune defenses and enable the uterus to preventor resolve infections. In fact, a long-acting PGF2 analogue, fenprostalene, injected some-time between days 7 and 10 postpartum reduced the incidence of endometritis in cows withdystocia and/or retained fetal membranes (Nakao et al., 1997). Fenprostalene should have in-creased uterine PGF2 production (Wade and Lewis, 1996), but the sampling frequency andsite of sample collection were not adequate to determine whether that was the case (Nakaoet al., 1997). Moreover, a single subcutaneous fenprostalene injection on the day of en-dometritis detection reduced the interval from parturition to conception (Nakao et al., 1997).Despite the evidence that exogenous PGF2 can promote uterine health in livestock, the com-plete mechanisms of action of exogenous PGF2 in uterine health have not yet been reported. If imposing treatments to increase uterine PGF2 and LTB4 production mitigates the im-munosuppressive effects of progesterone on uterine immune defenses and promotes uterinehealth in livestock environments, the mechanism for this is likely to be quite complex be-cause progesterone does more that just stimulate the production of immunosuppressantsand blocking factors. Progesterone also decreases the activity of a number of proinﬂamma-tory molecules and stimulates PGE synthase activity; PGE2 inhibits immune cell functionsin vitro (Segerson and Gunsett, 1993; Hunt et al., 1997; Hansen, 1998; Szekeres-Barthoet al., 2001; Arosh et al., 2002; Seals et al., 2002b). In addition, progesterone suppressesinterleukin (IL)-8 production, which stimulates chemotaxis, superoxide release, and gran-ule release from phagocytic cells, in reproductive tissues (Ito et al., 1994; Kelly et al., 1994;Mitchell et al., 2002, 2003; Loudon et al., 2003). Progesterone also suppresses the produc-tion of IL-6, which promotes B-cell differentiation and production of acute-phase proteins(Montes et al., 1995), and inhibits IL-12 production (Par et al., 2003). Interleukin-12 inducesinterferon- production and enhances natural killer-cell cytotoxicity, and PLA2 , presum-ably via increased free arachidonic acid, may mediate the effects of IL-12 (Par et al., 2003).Even though a variety of immunosuppressive effects have been ascribed to progesterone,the effects vary among reports, much of the research has been conducted with in vitro, andnot in vivo, models, and very little of the research has been conducted to understand therelationship between production of various proinﬂammatory molecules and the ability ofthe uterus in livestock to resistant or resolve infections.5. Conclusions Based on available literature, progesterone is the ovarian steroid that primarily governs theability of the uterus in livestock to resist infections. Progesterone typically suppresses im-
G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 291mune defenses and makes the uterus susceptible to infections. Injections of PGF2 increaseuterine production of PGF2 , and probably LTB4 . These two eicosanoids seem to promoteuterine immune defenses and uterine health. Eicosanoids and progesterone affect severalproinﬂammatory molecules that may modulate uterine immune defenses, but the roles ofthese molecules in determining whether the uterus is resistant or susceptible to infectionshave not yet been deﬁned. Determining how uterine PGF2 mitigates the immunosuppres-sive effects of progesterone and stimulates the uterus to resolve infections should clearly beimportant to scientists and clinicians working to understand the underlying causes of uterineinfections. This area of research should yield important new strategies for preventing andtreating uterine infections that do not include antibiotic and antimicrobial compounds.Acknowledgements The author is grateful for the contributions from his trainees and collaborators, partic-ularly R.P. Del Vecchio, I. Matamoros, A.A. Ramadan, R.C. Seals, S. Wang, and M.C.Wulster-Radcliffe.ReferencesArosh, J.A., Parent, J., Chapdelaine, P., Sirois, J., Fortier, M.A., 2002. Expression of cyclooxygenases 1 and 2 and prostaglandin E synthase in bovine endometrial tissue during the estrous cycle. Biol. Reprod. 67, 161–169.Arthur, G.H., Noakes, D.E., Pearson, H., 1989. Veterinary Reproduction and Obstetrics, sixth ed. Baillière Tindall, Philadelphia.Benoit, A.M., Dailey, R.A., 1991. Catheterization of the caudal vena cava via lateral saphenous vein in the ewe, cow, and gilt: an alternative to uteroovarian and medial coccygeal vein catheters. J. Anim. Sci. 69, 2971–2979.Binelli, M., Guzeloglu, A., Badinga, L., Arnold, D.R., Sirois, J., Hansen, T.R., Thatcher, W.W., 2000. Interferon-(modulates phorbol ester-induced production of prostaglandin and expression of cyclooxygenase-2 and phospholipase-A(2) from bovine endometrial cells. Biol. Reprod. 63, 417–424.Black, W.G., Ulberg, L.C., Kidder, H.E., Simon, J., McNutt, S.H., Casida, L.E., 1953. Inﬂammatory response of the bovine endometrium. Am. J. Vet. Res. 14, 179–183.Brinsﬁeld, T.H., Hawk, H.W., Righter, H.F., 1964. Interaction of progesterone and oestradiol on induced leucocytic emigration in the sheep uterus. J. Reprod. Fertil. 8, 293–296.Brinsﬁeld, T.H., Hawk, H.W., Leffel, E.C., 1967. Control by ovarian status of induced leukocytic responses in the sheep uterus. Am. J. Vet. Res. 28, 1723–1725.Cai, T.Q., Weston, P.G., Lund, L.A., Brodie, B., McKenna, D.J., Wagner, W.C., 1994. Association between neutrophil functions and periparturient disorders in cows. Am. J. Vet. Res. 55, 934–943.Chenault, J.R., McAllister, J.F., Chester, S.T., Dame, K.J., Kausche, F.M., 2001. Efﬁcacy of ceftiofur hydrochloride administered parenterally for ﬁve consecutive days for treatment of acute postpartum metritis in dairy cows. In: Proceedings of the 34th Annual Convention American Association of Bovine Practitioners, Vancouver, BC, pp. 137–138.Cooke, R.G., Ahmad, N., 1998. Delayed luteolysis after intra-uterine infusions of nordihydroguaiaretic acid in the ewe. Anim. Reprod. Sci. 52, 113–121.Del Vecchio, R.P., Matsas, D.J., Inzana, T.J., Sponenberg, D.P., Lewis, G.S., 1992. Effect of intrauterine bacterial infusions and subsequent endometritis on prostaglandin F2 metabolite concentrations in postpartum beef cows. J. Anim. Sci. 70, 3158–3162.Del Vecchio, R.P., Matsas, D.J., Fort´n, S., Sponenberg, D.P., Lewis, G.S., 1994. Spontaneous uterine infections are ı associated with elevated prostaglandin F2 metabolite concentrations in postpartum dairy cows. Theriogenology 41, 413–421.
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