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Immunology |
Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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circadian rhythm, immune, immunology, photoperiod, prolactin, prolactin receptor, sensitivity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in photoperiod result in robust alterations in circulating concentrations of prolactin (PRL) [7] and expression of its receptor (PRL-R) [3, 4] in the bovine. Although postpartum exogenous PRL administration does not appear to alter lactational performance in dairy cows [8], PRL has been implicated in changes of mammary function in the peripartum period. Sensitivity to PRL is also a potential mechanism for altered immune function. Peripheral lymphocytes express PRL-R mRNA in many species, including the bovine [9], and receptor expression in lymphocytes is reduced by PRL administration in rodents [10] and increased by bromocriptine treatment [3, 10]. We hypothesize that the sensitivity of an animal to shifts in PRL perceived by the differing expression of long and short forms of PRL-R with different photoperiod treatments is the mechanism by which the animal's immune system is influenced.
Two experiments were performed to test our hypothesis using in vitro and in vivo models to separate specific effects of photoperiod on PRL from nonspecific photoperiodic effects. The first objective of the present study was to determine the effect of exogenous PRL given via osmotic minipumps and daily injections on PRL concentrations, PRL-R mRNA expression on lymphocytes and cellular immune function in steers on different photoperiod treatments. In addition, we sought to determine the effect of adding PRL directly to the lymphocyte proliferation assay.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eight Holstein steer calves were housed in controlled-temperature indoor cubicles. After a week of pretreatment acclimation (12L:12D), steers were randomly assigned to either LDPP or SDPP. Lighting was provided by fluorescent lights at approximately 545 ± 15 lux at eye level of the steers. Lights came on at the same time for both treatment groups (0800 h) and went off at 1600 h for the SDPP steers and at 2400 h for the LDPP animals. After 1 wk of exposure to their assigned photoperiod treatment, exogenous PRL was administered to a subset of SDPP animals for 10 days via jugular vein injections or osmotic minipump implants (details below). After 10 days of PRL administration, animals remained on the photoperiod treatment for an additional 4 days. Steers were then switched to the next photoperiod/PRL treatment and acclimated for 1 wk before beginning the next round of PRL administration. This procedure was repeated until all animals received each of the four photoperiod/PRL treatments. Steers averaged 76 ± 4 days in age and 110.5 ± 5.4 kg in weight at the start of the experiment. Steers were individually fed a grain and alfalfa hay diet according to the guidelines of the National Research Council (1989) between 0830 and 1030 h each day. Adjustments were made every 2 wk for body weight gain and intake and refusals were recorded daily. Water was available to the steers at all times. All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee.
Implantation of Osmotic Minipumps
Osmotic minipumps were implanted while steers were standing in their stalls within the cubicle under moderate sedation (0.2 mg/kg Xylazine; Vedco, Inc., St. Joseph, MO). A small incision (2–3 cm) was made with a scalpel blade behind the shoulder on the animal's right side after subcutaneous application of 1 ml of local anesthetic (Lidocaine; Abbott Laboratories, Chicago, IL). A small subcutaneous pocket was created by blunt dissection with forceps and the Alzet osmotic minipump (2 ml capacity; 5.0 µl/h delivery rate; Durect Corporation, Cupertino, CA) was inserted such that the delivery portal faced the right hind leg at a downward angle. The incision was closed with skin staples and covered with iodophor paste (Prodine; Phoenix Pharmaceutical, Inc., St. Joesph, MO). Steers received 2 ml of a prophylactic antibiotic (Excenel; Pfizer, Kalamazoo, MI) 2 h before the implant procedure.
Exogenous Prolactin Administration
Treatments included SDPP, LDPP, SDPP plus daily intravenous injections of exogenous PRL (SDinj), and SDPP plus PRL administered via osmotic minipumps (SDom). The dose of PRL administered to SDPP animals was designed to achieve circulating PRL concentrations similar to those of LDPP animals. Recombinant methionyl bovine PRL for these experiments was obtained from Dr. John Byatt at Monsanto (Chesterfield, MO). Solutions of exogenous PRL were formulated so that animals received 4.0 mg/day rbPRL with 0.9% saline, administered either as three daily jugular vein injections (given at 0630, 1130, and 1700 h) or continuously via the minipumps (5.0 µl/h rate of delivery) over the course of 10 days. Because pattern of delivery can elicit divergent responses [11], we sought to compare multiple injections (i.e., pulsatile) to continuous release (i.e., chronic elevation). Three daily injections of PRL were chosen due to the relatively rapid clearance rates of PRL in dairy cows [12] and the lack of difference in circulating PRL concentrations seen in previous studies [13]. After the first treatment period, PRL was rapidly assayed to confirm the final circulating concentration of PRL.
Blood Sampling and Hormone Assays
Blood (10 ml) was collected from the jugular vein of steers restrained individually in their pens, using sterile Vacutainer tubes containing sodium heparin (Becton Dickinson and Co., Franklin Lakes, NJ). Collection occurred weekly during adjustment to photoperiod treatments and daily during the PRL administration periods, between 1030 and 1130 h, before the second injection of PRL. Samples for lymphocyte proliferation, real-time PCR, and neutrophil chemotaxis were taken at these times as well. Samples were placed on ice immediately after collection. Within 2 h of collection, plasma for hormone determination was obtained from whole blood after centrifugation (1850 x g, 20 min, 4°C) and stored at –20°C until assayed.
Plasma PRL concentrations were determined by radioimmunoassay as described by Miller et al. [14]. Mean intraassay and interassay coefficients of variation (two assays) were 5.6% and 10.6%, respectively. Assay sensitivity averaged 1.02 ng/ml.
Real-Time PCR
Bovine peripheral blood mononuclear cells (PBMCs) were used as the source of lymphocyte mRNA for real-time PCR. Bovine PBMCs were isolated from blood samples collected on sodium heparin by density gradient centrifugation through Histopaque-1077 (density: 1.077; Sigma, St. Louis, MO). The PBMCs were washed twice in RPMI-1640 (Sigma) and resuspended. The PBMCs were washed only once in RPMI before RNA isolation to minimize the degradation of RNA.
Real-time PCR was performed as described in Auchtung et al. [3]. Briefly, RNA was extracted from lymphocytes using Trizol reagent (Gibco BRL, Grand Island, NY) and stored at –80°C until further processing. Complementary DNA was used for real-time PCR. Taqman (Applied Biosystems, Foster City, CA) probes and primers were designed using Primer Express software for both the long- and short-form of PRL-R, and 18S rRNA was amplified as the internal reference. Detection was performed using an ABI PRISM 7700 Sequence Detector (Applied Biosystems) and validation was conducted according to Auchtung et al. [3]. Calculated input amounts were normalized to the 18S values. Final values are reported as expression values relative to calibrator cDNA (control mammary tissue) or as a percentage of baseline within treatment (pretreatment lymphocyte expression).
Lymphocyte Proliferation Assay
Lymphocyte proliferation assays were performed as described by Morrow-Tesch et al. [15] and Auchtung et al. [3]. Briefly, PBMCs were isolated from buffy coat by density gradient centrifugation and washed in RPMI. Concentration was adjusted to 5 x 106 cells/ml in RPMI supplemented with 10% fetal bovine serum (FBS; Sigma) and 50 µg/ml gentamycin (Sigma). Lymphocytes (100 µl) and mitogens (100 µl ConA [concanavalin A] and PWM [pokeweed mitogen]) were added in triplicate to 96-well flat-bottom sterile plates. Mitogens are proteins, often isolated from plants, that stimulate mitosis and proliferation of lymphocytes. Cells were incubated for 48 h at 37°C in 5% CO2. After incubation, 20 µl of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was added to each well and the cells were incubated for an additional 4 h, after which 100 µl of acidic isopropanol was added to each well, and the contents were mixed by repeated pipetting. The optical density (OD) of each well was measured within 1 h. Proliferation is expressed as the stimulation index (percentage of the unstimulated control), which is the OD of the treated wells/OD of the unstimulated control wells (cells receiving no mitogen).
In an additional study, rbPRL was added to the lymphocyte proliferation assay at various concentrations (0, 0.1, 1, 10, 100, 1000 µg/ml) to determine an optimum dose for comparison of proliferation among treatments. The optimum dose (1 µg/ml) was chosen such that the lymphocytes were responding on the steepest part of the dose-response curve, before the maximum response, to gain the most sensitivity of response. This dose was then added to wells with lymphocytes obtained from cows treated with LDPP, SDPP, or natural photoperiod. Mitogens (ConA: 0, 2, 20 µg/ ml; or LPS [lipopolysaccharide], 0, 1, 10, 50, 100 µg/ml) or control media were also added to the wells. Plates were incubated and read as described previously.
Neutrophil Chemotaxis Assay
Neutrophil chemotaxis was performed as described by Auchtung et al. [3]. Briefly, blood for neutrophil isolation was collected on EDTA and, following centrifugation, plasma, buffy coat, and one third of red blood cells were removed. Cold water was added to the remaining cells and mixed thoroughly. Isotonicity was restored after 1 min by adding 3x phosphate-buffered saline (PBS) and cells were centrifuged for 10 min at 475 x g. Cell pellets were resuspended in 2 ml PBS and neutrophil numbers determined using a Coulter counter (Coulter Electronics, Miami, FL). Cell concentration was adjusted to 3 x 106 cells/ml. The chemoattractants used were human interleukin-8 (IL-8; 100 ng/ml) and human complement 5a (C5a; 10–8 M). Approximately 30 µl of the chemoattractant or control media was placed in each well of the bottom chamber of the chemotaxis chamber. The adjusted cell solution was added to the top chamber at 50 µl per well and the chemotaxis chamber was incubated for 1 h. Following incubation, the noncell side of the filter was wiped with 3x PBS and then dried. The cell side was then dipped in methanol eight times and dried. Finally, the filter was stained with Diff-Quick (Fisher Scientific, Pittsburgh, PA), placed on a microscope slide, and allowed to dry. Five fields per well were counted to determine the number of neutrophils that migrated in response to either chemoattractant or media.
Statistical Analyses
Statistical analyses were performed using the SAS System, v. 8.2 (SAS Institute Inc., Cary, NC). A mixed model was used to analyze repeated-measures data, specifically comparing variables among photoperiod and PRL treatments and across time. Temperature and pretreatment hormone values were used as covariates in the model analyzing PRL concentrations. Treatment least-squares means and standard error of the difference (SED) are reported.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Over the course of the experiment, all steers maintained good health. Total intake (grain and alfalfa hay cubes) did not differ among the treatments (P = 0.77). Body weight increased, on average, from 110.5 ± 5.4 kg at the start of the study to 180.3 ± 7.8 kg at the conclusion of the study and was not different among the photoperiod or PRL treatments (P = 0.56).
Prolactin and Prolactin Receptor
Concentrations of PRL did not differ on Day 0 of the experiment (P = 0.43) before photoperiod or PRL treatments began. Within the first week of photoperiod treatment, plasma PRL concentrations were greater (P = 0.03) in animals on LDPP relative to SDPP animals. Concentrations during the first week of photoperiod treatment averaged 10.2 and 5.3 ng/ml for LDPP and SDPP animals, respectively. During the 10-day PRL treatment period, steers receiving exogenous PRL via osmotic minipump (SDom) had PRL concentrations similar to steers receiving PRL via daily injections (SDinj) and LDPP steers (Fig. 1). Prolactin concentrations were lower (P < 0.05) in steers exposed to SDPP only compared with all other treatment groups during the PRL-treatment period.
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Expression of PRL-R mRNA in lymphocytes of Holstein steers did not differ before photoperiod or PRL treatment (P = 0.57). However, SDPP treatment increased PRL-R mRNA expression (P = 0.02) relative to LDPP treatment within the first week of photoperiod treatment, before PRL treatment. During the PRL-treatment period, long-form PRL-R mRNA expression was similar among LDPP, SDom, and SDinj treatments (Fig. 2) and significantly greater in SDPP-treated animals. There was no difference in PRL-R mRNA expression due to delivery method, i.e., injections vs. pump. The short form of PRL-R mRNA followed similar trends to the long form of the receptor; however, there were no significant differences among treatment groups.
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Lymphocyte Proliferation and Neutrophil Chemotaxis
Lymphocyte proliferation in response to ConA and PWM was greater for animals treated with SDPP than LDPP (P < 0.05) before PRL treatment. Following PRL treatment, lymphocyte proliferation of SDom and SDinj animals decreased to levels similar to LDPP animals (Fig. 3), which was significantly less than SDPP animals.
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Prior to PRL treatment, neutrophil migration toward both IL-8 and C5a were greater for SDPP steers than LDPP (P < 0.05). Neutrophil chemotaxis was decreased following PRL treatment for SDom- and SDinj-treated animals compared with SDPP treatment alone (Fig. 4).
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Proliferation in Response to Prolactin
Addition of PRL to the lymphocyte proliferation assay showed dose responsiveness (P = 0.04). The dose of PRL selected as optimal (1 µg/ml) for use in the lymphocyte proliferation assay elicited a submaximal response. It was selected because we were interested in comparing relative sensitivity to PRL among treatments rather than maximal stimulation. Proliferative response of lymphocytes to PRL alone was greater in SDPP animals compared with LDPP animals or control animals (Fig. 5), although there was no difference between the LDPP or control animals. Addition of PRL with either mitogen, ConA or LPS, enhanced lymphocyte proliferation (P < 0.05) for SDPP-treated animals compared with the stimulation of SDPP lymphocytes with only the mitogen but did not significantly alter the proliferation of lymphocytes from LDPP animals. Lymphocyte proliferation with the addition of PRL to the mitogens was greater (P < 0.05) in SDPP animals relative to LDPP animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because our intent was to increase circulating PRL concentration similar to that of LDPP animals, we took care that the amount of PRL delivered from the osmotic minipump during the 10 days of PRL treatment was enough to raise the plasma PRL of the steers for the entire time, giving us a chronic increase in PRL. Barrington et al. [13] noted that single daily injection of rbPRL was insufficient to increase serum PRL concentrations, possibly due to the rather rapid clearance of PRL from the system. Therefore, we adjusted our treatment to thrice-daily injections. With this, we tried to accomplish an acute increase of PRL in a pulsatile manner so as to compare the chronic elevation of PRL via osmotic minipump to the acute elevation obtained with daily injections.
Interestingly, no difference was observed with respect to the mode of PRL delivery. Given the time of blood sample collection relative to injection, we expected the PRL-injected animals to have cleared the exogenous PRL at the time of sampling and had circulating PRL concentrations similar to those of noninjected animals. Yet circulating PRL concentrations did not differ between the PRL-injected and chronically infused animals. The reason for the extended period of elevated PRL in injected steers is unclear, although it may relate to age or reproductive status of the animals, as we have evidence that lactating dairy cows rapidly clear PRL injections (unpublished results). Further investigation of the dynamics of PRL clearance would be warranted. Regardless, the end result of the increased PRL, i.e., decreased PRL-R expression and decreased cellular immune function relative to animals with lower PRL concentrations, did not differ between the two exogenous PRL-delivery methods.
The decrease in PRL-R mRNA expression in lymphocytes following PRL administration is consistent with Di Carlo et al. [10]. In that study, rats were injected with ovine PRL and PRL-R mRNA expression was subsequently estimated in peripheral lymphocytes. The inverse relationship of decreased PRL-R mRNA expression following increased PRL is similar to our observations of increased PRL and decreased receptor mRNA expression in animals on LDPP. In previous studies, both long and short forms of PRL-R have shown significant differences between photoperiod treatments [3, 4]. In this study, the short form followed a similar pattern to the long form of the receptor, although there was not a significant difference among treatments. Given that the short form of the receptor tends to change less dramatically than the long form, the lack of significance could be due to the small sample numbers. Physiologically, however, the effect on the long form is perhaps most relevant to the observation that immune function shifted in response to PRL and photoperiod treatments, as the expression of the long form was higher in SDPP animals relative to LDPP animals.
The reduction of lymphocyte proliferation and neutrophil chemotaxis after administration of exogenous PRL observed here is consistent with reports in humans [16] and other species [17]. In addition, there are reports that elevated concentrations of PRL are found in patients with autoimmune diseases and reduction of elevated circulating concentrations of PRL is beneficial to the overall health of those individuals [18]. These data are in contrast with studies in humans that observed a transient decrease in responsiveness of peripheral lymphocytes to mitogen stimulation following a decrease in PRL concentrations [19]. However, it should be noted that those patients were chronically ill and decreases observed in their PRL concentrations were acute and may not have been directly related to basal levels. Additional research is needed to determine the extent to which circulating PRL is critical to immune function.
Stimulation of lymphocytes with PRL in vitro has been shown to increase proliferation significantly [20]. However, most studies have used a dose much higher than that used in the present study. Whereas the addition of PRL to lymphocytes of control or LDPP-treated animals in this study did elicit a response above baseline, it was not significant. In contrast, the addition of PRL to lymphocytes of SDPP-treated animals did result in a significant increase in proliferation. This suggests that the increase in PRL-R on lymphocytes of SDPP animals, relative to LDPP-treated animals, increases the ability of PRL to stimulate lymphocyte proliferation, potentially improving cellular immune function of those animals.
Prolactin has been implicated in suppression of apoptosis [21] and is particularly beneficial under conditions of increased circulating glucocorticoids, such as the stress around the time of parturition, when PRL has been observed to maintain function and survival of lymphocytes [22]. Conversely, there is also evidence that administration of pharmacological doses of PRL decreases survival of septic mice [23]. Whereas photoperiod manipulation alters PRL concentrations and expression of PRL-R mRNA, photoperiod treatment does not elicit changes in cortisol concentrations in cattle [24], suggesting an uncoupling of PRL and glucocorticoids. This may offer an explanation to differences seen in cellular immune function among species when PRL is introduced into the system.
In a previous study [3], we observed that LDPP animals treated with bromocriptine to reduce circulating PRL concentrations increased lymphocyte proliferation and neutrophil chemotaxis and decreased PRL-R mRNA expression in lymphocytes in Holstein steers relative to LDPP treatment alone. Those observations support the hypothesis that shifts in circulating PRL and PRL-R expression is the mechanism whereby photoperiod manipulation affects changes in select measures of cellular immune function. In the present study, administration of exogenous PRL increased circulating PRL and decreased expression of the long form of PRL-R in SDPP-treated animals. Relative to animals treated with SDPP only, administration of exogenous PRL decreased lymphocyte proliferation and neutrophil chemotaxis, similar to LDPP. These data support our hypothesis that increased sensitivity and responsiveness to PRL may mediate the enhancement of cellular immune function observed in animals treated with short-day photoperiod.
The results of these studies may have practical implications for animal management, not only in cattle but in other domestic and nondomestic species as well. Manipulation of PRL, and therefore PRL sensitivity, with photoperiod is only one potential environmental approach to affect such a response. Other physical (e.g., heat) and psychological (e.g., grouping) stressors are known to impact PRL secretion and thus may affect immune function. Greater understanding of the mechanisms that underpin interactions of PRL and animal health may therefore support noninvasive management techniques to optimize animal health and performance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Geoffrey E. Dahl, Department of Animal Sciences, 230 Animal Sciences Laboratory, MC-630, 1207 W. Gregory Dr., University of Illinois, Urbana, IL 61801. FAX: 217 333 7088; gdahl{at}uiuc.edu
Received: 15 April 2004.
First decision: 17 May 2004.
Accepted: 22 July 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  G. E. Dahl Effects of short day photoperiod on prolactin signaling in dry cows: A common mechanism among tissues and environments? J Anim Sci, March 1, 2008; 86(13_suppl): 10 - 14. [Abstract] [Full Text] [PDF]
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= P < 0.05 within photoperiod treatments within mitogen). Standard error of the difference = 89.7