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Partial characterization of immunosuppressive proteins from bovine uterine luminal secretions
THERIOGENOLOGY
PARTIAL CHARACTERIZATIONOF IMMUNOSUPPRESSIVE PROTEINS FROM BOVINE UTERINE LUMINAL SECRETIONS E. C. Segerson, Jr.1 D. C. Stephensonl, P. L. Matterson 1, D. W.'Libbyl T. R. Hansen* and R. D. Randel3 Department of Animal Sciencel, North Carolina A&T State University, Greensboro, NC 27411 and Texas A&M Universit Agricultural Research and Extension Center2 , Overton, TX 75684 Received for publication: August 30, 1983 Accepted: ApriZ 16, 1986 ABSTRACT Unfractionated and fractionated uterine luminal protein (ULP) secretions collected from nonpregnant and pregnant beef cows on Day 17 post-breeding were tested in vitro for suppression of lymphocyte blastogenesis to the mitogen phytohemagglutinin (PHA). In replicated experiments, ULP from nonpregnant and pregnant cows was separated into five molecular weight (Mr) fractions with Sephacryl S-200. Unfractionated (25 to 400 pg/ml) and fractionated (25 to 100 pg/ml) ULP was added to cultures containing 5 x lo5 bovine lymphocytes and 0.4 11sof PHA in a complete culture medium. At 48 hr, 0.5 PCi of %i-thymidinewas added to cultures, and cells were harvested at 60 h by automation. Thymidine incorporation data were expressed as percentage of control (no ULP) values. Unfractionated and all S-200 ULP fractions from nonpregnant and pregnant cows suppressed (PcO.05 to 0.001) lymphocyte blastogenesis to PHA, but to varying degrees of suppression. Unfractionated ULP was more suppressive (PcO.05) for pregnant than nonpregnant cows, which was likely due to the greater (PcO.05) immunosuppressive activity of S-200 fractions I (>219,000 Mr) and V (-14,000 Mr) from the pregnant cows. At 25 pg ULP/ml, mean (* SEM) % of control values for fraction I from pregnant and nonpregnant cows were 9.1 _t 3.3 and 36.6 f 8.5%, respectively (PcO.05). Values for fraction V were 15.7 + 6.5 and 46.6 + b-l%, respectively (P
Acknowledgments Research was supported in part by CSRS/USDA Grant No. NCX-045-580-120-l to E. C. Segerson. Journal paper TA 20971, Texas Agr. Exp. Station. JUNE 1986 VOL. 25 NO. 6
767
THERIOGENOLOGY
INTRODUCTION
Investigations in cows (1,2) and ewes (3-5) have demonstrated that the immune system within the uterus is capable of responding to antigenic material; this suggests that immunological adjustments are necessary for maintenance of the conceptus allograft. In vitro immunological test systems have demonstrated that various macromolecules, recovered from the uterine,ilumenduring pre- and peri-implantationperiods, may be involved in providing protection to the conceptus through modification of maternal cell-mediated immune responses. Suppressed response of maternal T-lymphocytes to the mitogen phytohemagglutinin(PHA) has been demonstrated for uterine luminal protein (ULP) secretions from nonpregnant and pregnant swine (6,7), nonpregnant (8) and pregnant (8-10) sheep and nonpregnant (11,12) and pregnant (11-13) beef cattle. During the late luteal phase of the cycle or early pregnancy, ULP secretions were more suppressive for pregnant than nonpregnant ewe,?(3) and beef cows (11-13). Thus far, immunosuppressiveactivity has been attributed to proteins of molecular weight (Mr) of -15,000 for gilts (6,14,15) and 17,400 and 42,600 for ewes (16). The characterizationof bovine immunosuppressiveULP has not been reported. Therefore, this report presents initial data on the characterizationof bovine immunosuppressiveproteins from unfractionated ULP previously shown to suppress lymphocyte blastogenesis in mitogenstimulated and mixed lymphocyte cultures (11).
MATERIALS AND METHODS
ULP secretions collected from nonpregnant and pregnant Angus and Brahman cows on Day 17 post-breedingwere used for the investigation. Procedures for collecting, processing and storing of the ULP preparations have been described (17). These secretions had been frozen in buffer for -4 mo prior to this investigation. Day 17 was chosen for immunosuppressiveinvestigationsbecause a previous report (13) indicated that Day 17 was the earliest day after mating in which bovine ULP exerted immunosuppressiveactivity. These secretions were previously characterized for qualitative and quantitative protein components (17) and were subjected to in vitro testing of the unfractionated preparations for immunosuppressive activity (11). For this investigation,aliquots of dialyzed protein were combined from nonpregnant Angus (n=5) and Brahman (n=3) and pregnant Angus (n=8) and Brahman (n=5) cows. ULP preparations were combined according to pregnancy status in order to provide enough protein for the immunosuppressiveexperiments requiring both unfractionated and fractionated preparations.
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ULP (-25 mg) preparations from nonpregnant and pregnant cows were fractionated by gel filtration chromatography using Sephacryl s-200 (column dimensions = 142.2 x 1.5 cm). The eluting buffer consisted of 0.05 M phosphate-bufferedsaline containing 0.02% NaN3 at pH 7.4. Two-ml fractions were collected and the flow rate The S-200 column was calibrated of the column was 15.0 ml/h. with the following Mr standards: bovine y-globulin (150,000), bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (31,000) and cytochrome c (12,500). Sephacryl S-200 and protein standards were obtained from Sigma Chemical Co., St. Louis, MO. The logarithm of the Mr of each standard was used for t e calculation of the regression equation, y = 6.823 0.0132~ (R3 = 0.98, P
Lymphocyte blastogenesis experiments were conducted for unfractionated and fractionated ULP samples representing nonpregnant and pregnant cows. For fractionated ULP samples, five major fractions representing S-200 peaks were tested for immunosuppressive activity. Procedures for bovine lymphocyte collection and extraction techniques and the in vitro lymphocyte-PHAculture experiments have been described (11-13). Briefly, immunosuppressive activity was evaluated in sterile Falcon Micro Test III plates in which each well contained 5 x 105 bovine lymphocytes, 0.4 pg of the T-lymphocyte specific mitogen PHA (Wellcome Research Labs., Beckenham, England) in 4 ~1 of distilled water (except for one control culture),and unfractionated ULP (25 to 400 pg/ml) and S-200 ULP fractions (25 to 100 pg/ml) of varying concentrations in quadruplicate cultures. ULP was added to culture wells -10 min after the addition of lymphocytes and PHA. In a preliminary experiment, 0.4 pg of PHA yielded maximal stimulation of 5 x lo5 lymphocytes without altering their viability. Control cultures (two types) contained lymphocytes only or lymphocytes plus PHA. Cultures were incubated in humidified 95% air:5% CO2 at 37.5 C. At 48 h of culture, 0.5 PCi of 3Hthymidine (3H-3 methyl, 6.7 Ci/mmol; New England Nuclear, Boston, MA) was added to each culture well (excluding viability cultures) in 0.05 ml of complete culture medium (RPMI-1640 containing 10% fetal bovine serum, 2% extra 200 mM glutamine (v/v), 100 IU of penicillin and 100 ug of streptomycin/ml),whichyielded a total volume of 0.2 ml/culture well. Cultures were harvested with 10% trichloroaceticacid at 60 f 1 R on cellulose-triacetatefilter
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discs using an automatic cell harvester (Skatron, Inc., Sterling, VA). Discs were placed in vials containing 8 ml of scintillation fluid and counted (8). For cultures involving the incorporation of 'H-thy&dine, parallel cultures were used to determine percentage viability of lymphocytes at the onset and termination of culture using the trypan blue exclusion method. A detailed description has been reported (9). For statistical analyses, counts per minute (cpm) for all cultures receiving 3H-thymidine were converted to picomoles (pmol) of thymidine according to a standard formula (19). To standardize immunosuppressivedata among experiments, thymidine incorporation data in test protein cultures were expressed as percentages ('$) of control (lymphocytes+ PHA) incorporation data using the following formula: 100 x (pm01 of thymidine incorporation in test protein culture-background)/(pm01 of thymidine incorporation in control culture-background). Thymidine incorporation (percent of control) and viability data were subjected to regression analysis. Individual regression lines were constructed from prediction equations in which protein concentrationwas the independent variable and percent of control and percent of viability were dependent variables. Significant R2 values reflected suppression of PHAinduced lymphocyte blastogenesis or reduced lymphocyte viability. Differences in degree of immunosuppressiveactivity at specific ULP concentrations for unfractionated ULP samples from nonpregnant and pregnant cows and among S-200 fractions within and between reproductive stages were assessed by analysis of variance considering concentration and S-200 fractions as main effects. Mean values for degree of suppression at a given concentration between pregnant and nonpregnant fractions or among S-200 fractions within a pregnancy status were separated by Duncan's multiple range test (when applicable). All analyses have been described (20). Additionally, quantitative estimates of the relative size of each fraction comprising the S-200 profiles for ULP from nonpregnant and pregnant cowswere derived by cutting out each fraction (in duplicate determinations)from the tracing paper and expressing the weight of each fraction as a percentage of the weight of the entire profile. Limited observations did not warrant a thorough statistical evaluation.
RESULTS Unfractionated Day 17 ULP from nonpregnant and pregnant beef cows suppressed (P
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nonpregnant and pregnant cows, mean percent of control values for 100 and 200 e ULP/ml were less (P~0.05) for pregnant than nonpregnant cows, thus reflecting greater immunosuppressive activity for ULP from pregnant cows. For all experiments in this investigation,mean (k SEM) pmol of thymidine for cultures containing lymphocytes alone or lymphocytes plus PHA were 1.6 f 0.2 and 24.9 + 2.2, respectively. A representative S-200 profile for fractionated ULP samples from nonpregnant and pregnant cows is illustrated (Figure 2). Five major fractions were revealed from gel filtration chromatography and the M, estimates appear above each major fraction. Protein M, estimates ranged from 5,700 to >248,000 (void volume). Therefore, fraction I may contain protein(s) of much greater Mr than 248,000. Although fractionated samples were few in number, the percentage of total protein in each fraction appeared to be similar between nonpregnant and pregnant cows, and the percentages for fractions I, II, III, IV and V were 10.7 and 7.1X, respectively 26.2 and 23.'7",respectively; 30.0 and 28.7%, respectively; 16.5 and 26.4%, respectively; and 16.7 and 14-l%, respectively.
UNFRACTIONATED PROTEIN
. . \ . % . \ .
Nonpregnant Pregnant -
SO
PROTEIN Figure 1.
100
-
150
-
\
-
260
250
360
CONCENTRATION,.ug/ml
Regression lines constructed from prediction equations illustrating the effect of bovine ULP upon PHA-stimulated lymphocyte blastogenesis. Protein secretions were collected on Day 17 of pregnancy and the estrous cycle.
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THERIOGENOLOGY
I
v I
looi
1
120
I
140
ELUTION Figure 2.
160
l60
220
200
240
VOLUME,ml
Representativegel (Sephacryl S-ZOO) filtration profile of bovine ULP collected on Day 17 of pregnancy and the estrous cycle.
Immunosuppressivedata for S-200 ULP fractions from nonpregnant cows are presented (Figure 3). All five fractions suppressed (P~O.001) lymphocyte blastogenesis to PHA. Regression equations for the individual fractions follow: I (Y = 79.3 1.039x, R2 = 0.69, P
Immunosuppressivedata for S-200 ULP fractions from pregnant cows are presented (Figure 4). All five fractions suppressed (PiO.05 to 0.001) lymphocyte blastogenesis to PHA. Regression equations for the individual fractions follow: I (y = 87.7 2.225x, R2 = 0.82, P
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THERIOGENOLOGY
PROTEIN Figure 3.
CONCENTRATION,ug/ml
Regression lines constructed from prediction equations illustrating the effect of Sephacryl S-200 fractions (I to V) of bovine ULP (Day I_7 of the estrous cycle) upon PHA-stimulated lymphocyte blastogenesis.
P
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PROTEIN Figure 4.
CONCENTRAT\ON.ug/mI
Regression lines constructed from prediction equations illustrating the effect of Sephacryl S-200 fractions (I to V) of bovine ULP (Day 17 of pregnancy) upon PHA-stimulated lymphocyte blastogenesis.
respectively (PO.l). At the termination of the culture periods, mean (+ SEM) percent viabilities for combined control (lymphocytes plus PHA) and combined ULP cultures, irrespective of pregnancy status, were 59.2 * 2_O%(range of 55.0 to 64.0%) and 58.4 -C1.9% (range of 21.4 to 80.0%), respectively.
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FRACTION
100
I
Nonpregnan t Pregnant -
FRACTION
V
Nonpregnan t Pregnant - -
-
-
80
60
I 20
40
60
PROTEIN
Figure 5.
, 80
CONCENTRATION,
2-o
4’0
6.0
80
ug/ ml
Comparison of degree of immunosuppressiveactivity of Sephacryl S-200 ULP fractions I and V (high and low molecular weight, respectively) for cows on Day 17 of pregnancy and the estrous cycle.
DISCUSSION The degrees of immunosuppressiveactivity of Day 17 ULP secretions from nonpregnant and pregnant beef cows in this investigation were similar to the degrees of immunosuppressive activity previously reported for these preparations (11). These data further support previous data indicating that ULP secretions collected on Day 14 from ewes (8) and on Days 17 to 18 from beef cows (11-13) are more immunosuppressivein pregnant than in nonpregnant ewes and beef cows. Data in this study demonstrate that the majority of immunosuppressiveactivity resulted from at least two proteins, one of relatively low M, (-14,000) and the other considered to be of high M, (>219,000). These findings have not been previously reported for beef cattle. The low M, protein is similar in M, to the principal immunosuppressiveprotein (-15,000) previously reported for swine (6) and rabbits (21). At this time it is unknown whether more than one immunosuppressiveprotein is present in S-200 fractions I and V.
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Greater immunosuppressiveactivity of bovine ULP secretions for pregnant than nonpregnant cows, as demonstrated in this and other studies (U-13), is likely due to greater quantities, activation or numbers of immunosuppressiveproteins in S-200 fractions I (high M,) and V (low M,). Although percentage composition of fractions I and V was not greater for pregnant than nonpregnant cows, perhaps the immunosuppressiveproteins constituted a minimal percentage of total protein within each fraction. Further, although mean quantities of total ULP were identical between pregnant and nonpregnant cows (18.9and 18.9 mg, respectively) on Day 17, polypeptides of 9,000 and 15,000 Mr were quantitatively greater in ULP secretions of pregnant than nonpregnant cows (17). In another study (22), three high M, polypeptides (range of 306,800 to 342,800 M,) were present in uterine flushings on Day 19 only for pregnant cows. In this study, there were no differences in percent of control values between corresponding fractions II, III and IV for nonpregnant and pregnant cows, thus supporting the postulation that greater immunosuppressiveactivity of ULP for pregnant than nonpregnant cows results from low (14,000 and high (>219,000) Mr proteins. The low and high M, immunosuppressiveproteins identified in this investigation appear to be derived primarily from endometrial tissue; however, the conceptus may also be responsible for part of the production of the immunosuppressiveproteins. It is conceivable that the presence of the conceptus could either directly or indirectly influence the synthesis or secretion or both of the low and high M, immunosuppressiveproteins, since immunosuppressive activity of these proteins was greater in ULP secretions from pregnant than nonpregnant cows. Viability data indicate that immunosuppressiveactivity of ULP was probably not mediated by overall lymphocyte cytotoxicity, although one must consider that the viability of a specific subpopulation(s) of T-lymphocytes responsible for the suppressor activity in ULP cultures may have declined while another subpopulation(s) proliferated. Viability data are analogous to viability data previously reported for immunosuppressivestudies in beef cattle (ll-13), swine (6) and sheep (8,9). Further, previous mixed lymphocyte culture (MLC) data (11) indicate that immunosuppressionof the ULP preparations used in this investigation was not mediated by inhibition of binding of PHA to the lymphocytes. MLC data in that study (11) indicated that ULP secretions were more suppressive for pregnant than nonpregnant cows, similar to immunosuppressiveactivities observed for experiments utilizing PHA. At the termination of lymphocyte cultures containing PHA and ULP in this investigation, aggregations of lymphocytes were similar to those in control samples with PHA alone. If PHA were unable to interact with lymphocyte binding sites, aggregation of lymphocytes would seem unlikely.
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REFERENCES
1.
Rowson, L. E. A., Lamming, G. E. and Fry, R. N. The relationship between ovarian hormones and uterine infection. Vet. Rec. -65:333-340 (1953).
2.
Black, W. G., Simon, J., Kidder, H. E. and Wiltbank, J. N. Bactericidal activity of the uterus in the rabbit and the cow. Am. J. Vet. Res. -15:247-251 (1954).
3.
Hawk, H. W., Turner, G. D. and Sykes, J. F. Variation in the inflammatory and bactericidal activity of the sheep J. Vet. Res. 22: uterus during the estrous cycle. Am. 689-692 (1961).
4.
Reimers, T. J. and Dziuk, P. J. The survival of intrauterine skin autografts and allografts in sheep. J. Reprod. Fertil. -38:465-467 (1974).
5.
Segerson, E. C. and Libby, D. W. Mitogenic response of lymphocytes collected from jugular and uterine veins and the uterine lumen of estrous, day 14 and ovariectomized ewes. Biol. Reprod. -30:126-133 (1984).
6.
Murray, F. A., Segerson, E. C. and Brown, F. T. Suppression of lymphocytes in vitro by porcine uterine secretory protein. Biol. Reprod. -19:15-25 (1978).
7.
Murray, F. A., Zurcher, V. and Grifo, A. P. Suppression of lymphocyte reactivity in vitro by porcine allantoic and amniotic fluids. Theriogenology-11:217-225 (1979).
8.
Segerson, E. C. Immunosuppressiveeffect of ovine uterine secretory protein upon lymphocytes in vitro. Biol. Reprod. -25377-84 (1981).
9.
Segerson, E. C., Moffatt, R. J., Bazer, F. W. and Roberts, R. M. Suppression phytohemagglutinin-stimulatedlymphocyte blastogenesis by ovine uterine milk protein. Biol. Reprod. -30:1175-1186 (1984).
10.
Staples, L. D., Brown, D., Binns, R. M. and Heap, R. B. The influence of protein hormones and conceptus extracts on sheep lymphocyte transformation induced in vitro. Placenta 4:125131 (1983).
11.
Segerson, E. C., Libby, D. W., Getz, W. R. and Randel, R. D. Immunosuppressiveeffect of uterine secretory protein from Angus and Brahman cows upon lymphocytes in vitro. J. Anim. Sci. 2:1047-1059 (1984).
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12.
Fisher, S. J., Gimenez, T. and Henricks, D. M. Immunosuppressive activity associated with early pregnancy in the bovine. Biol. Reprod. -32:894-906 (1985).
13.
Roberts, G. P. Inhibition of lymphocyte stimulation by bovine uterine proteins. J. Reprod. Fertil. -50:337-339 (1977).
14.
Murray, F. A., Bazer, F. W., Wallace, H. D. and Warnick, A. C. Quantitative and qualitative variation in the secretion of protein by the porcine uterus during the estrous cycle. Biol. Reprod. 13314-320 (1972).
15.
Squire, G. D., Bazer, F. W. and Murray, F. A. Electrophoretie patterns of porcine uterine protein secretions during the estrous cycle. Biol. Reprod. 1:321-325 (1972).
16.
Staples, L. D. Isolation and partial characterizationof an antigen associated with pregnancy in the ewe. Biol. Reprod. -22:675-685 (1980).
17.
Segerson, E. C., Hansen, T. R., Libby, D. W., Randel, R. D. and Getz, W. R. Ovarian and uterine morphology and function in Angus and Brahman cows. J. Anim. Sci. -59:1026-1046 (1984).
18.
Lowry, 0. H., Rosebrough, N. J,, Farr, A. L. and Randall, R. J_ Protein measurement with the Folin phenol reagent. J. Biol. Chem 193:265-275 (1951).
19.
Burford-Mason, A. P. and Gyte, G. M. L. An alternative method of expressing results of lymphocyte transformation experiments. J. Immunol. Methods 28:391-394 (1979).
20.
Snedecor, G. W. and Cochran, W. G. Statistical Methods. Iowa State University Press, Ames, 1967, pp. 59-62, 135-171 and 274.
21.
Mukherjee, A. B., Ulane, R. E. and Agrawal, A. K. Role of uteroglobin and transglutaminasein masking the antigenicity of implanting rabbit embryos. Am. J. Reprod. Immunol.2:135141 (1982).
22.
Bartol, F. F., Thatcher, W. W., Bazer, F. W., Kimball, F. A., Chenault, J. R., Wilcox, C. J. and Roberts, R. M. Effects of the estrous cycle and early pregnancy on bovine uterine, luteal and follicular responses. Biol. Reprod. 25:759-776. (1981).
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PARTIAL CHARACTERIZATIONOF IMMUNOSUPPRESSIVE PROTEINS FROM BOVINE UTERINE LUMINAL SECRETIONS E. C. Segerson, Jr.1 D. C. Stephensonl, P. L. Matterson 1, D. W.'Libbyl T. R. Hansen* and R. D. Randel3 Department of Animal Sciencel, North Carolina A&T State University, Greensboro, NC 27411 and Texas A&M Universit Agricultural Research and Extension Center2 , Overton, TX 75684 Received for publication: August 30, 1983 Accepted: ApriZ 16, 1986 ABSTRACT Unfractionated and fractionated uterine luminal protein (ULP) secretions collected from nonpregnant and pregnant beef cows on Day 17 post-breeding were tested in vitro for suppression of lymphocyte blastogenesis to the mitogen phytohemagglutinin (PHA). In replicated experiments, ULP from nonpregnant and pregnant cows was separated into five molecular weight (Mr) fractions with Sephacryl S-200. Unfractionated (25 to 400 pg/ml) and fractionated (25 to 100 pg/ml) ULP was added to cultures containing 5 x lo5 bovine lymphocytes and 0.4 11sof PHA in a complete culture medium. At 48 hr, 0.5 PCi of %i-thymidinewas added to cultures, and cells were harvested at 60 h by automation. Thymidine incorporation data were expressed as percentage of control (no ULP) values. Unfractionated and all S-200 ULP fractions from nonpregnant and pregnant cows suppressed (PcO.05 to 0.001) lymphocyte blastogenesis to PHA, but to varying degrees of suppression. Unfractionated ULP was more suppressive (PcO.05) for pregnant than nonpregnant cows, which was likely due to the greater (PcO.05) immunosuppressive activity of S-200 fractions I (>219,000 Mr) and V (-14,000 Mr) from the pregnant cows. At 25 pg ULP/ml, mean (* SEM) % of control values for fraction I from pregnant and nonpregnant cows were 9.1 _t 3.3 and 36.6 f 8.5%, respectively (PcO.05). Values for fraction V were 15.7 + 6.5 and 46.6 + b-l%, respectively (P
Acknowledgments Research was supported in part by CSRS/USDA Grant No. NCX-045-580-120-l to E. C. Segerson. Journal paper TA 20971, Texas Agr. Exp. Station. JUNE 1986 VOL. 25 NO. 6
767
THERIOGENOLOGY
INTRODUCTION
Investigations in cows (1,2) and ewes (3-5) have demonstrated that the immune system within the uterus is capable of responding to antigenic material; this suggests that immunological adjustments are necessary for maintenance of the conceptus allograft. In vitro immunological test systems have demonstrated that various macromolecules, recovered from the uterine,ilumenduring pre- and peri-implantationperiods, may be involved in providing protection to the conceptus through modification of maternal cell-mediated immune responses. Suppressed response of maternal T-lymphocytes to the mitogen phytohemagglutinin(PHA) has been demonstrated for uterine luminal protein (ULP) secretions from nonpregnant and pregnant swine (6,7), nonpregnant (8) and pregnant (8-10) sheep and nonpregnant (11,12) and pregnant (11-13) beef cattle. During the late luteal phase of the cycle or early pregnancy, ULP secretions were more suppressive for pregnant than nonpregnant ewe,?(3) and beef cows (11-13). Thus far, immunosuppressiveactivity has been attributed to proteins of molecular weight (Mr) of -15,000 for gilts (6,14,15) and 17,400 and 42,600 for ewes (16). The characterizationof bovine immunosuppressiveULP has not been reported. Therefore, this report presents initial data on the characterizationof bovine immunosuppressiveproteins from unfractionated ULP previously shown to suppress lymphocyte blastogenesis in mitogenstimulated and mixed lymphocyte cultures (11).
MATERIALS AND METHODS
ULP secretions collected from nonpregnant and pregnant Angus and Brahman cows on Day 17 post-breedingwere used for the investigation. Procedures for collecting, processing and storing of the ULP preparations have been described (17). These secretions had been frozen in buffer for -4 mo prior to this investigation. Day 17 was chosen for immunosuppressiveinvestigationsbecause a previous report (13) indicated that Day 17 was the earliest day after mating in which bovine ULP exerted immunosuppressiveactivity. These secretions were previously characterized for qualitative and quantitative protein components (17) and were subjected to in vitro testing of the unfractionated preparations for immunosuppressive activity (11). For this investigation,aliquots of dialyzed protein were combined from nonpregnant Angus (n=5) and Brahman (n=3) and pregnant Angus (n=8) and Brahman (n=5) cows. ULP preparations were combined according to pregnancy status in order to provide enough protein for the immunosuppressiveexperiments requiring both unfractionated and fractionated preparations.
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JUNE 1986 VOL. 25 NO. 6
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ULP (-25 mg) preparations from nonpregnant and pregnant cows were fractionated by gel filtration chromatography using Sephacryl s-200 (column dimensions = 142.2 x 1.5 cm). The eluting buffer consisted of 0.05 M phosphate-bufferedsaline containing 0.02% NaN3 at pH 7.4. Two-ml fractions were collected and the flow rate The S-200 column was calibrated of the column was 15.0 ml/h. with the following Mr standards: bovine y-globulin (150,000), bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (31,000) and cytochrome c (12,500). Sephacryl S-200 and protein standards were obtained from Sigma Chemical Co., St. Louis, MO. The logarithm of the Mr of each standard was used for t e calculation of the regression equation, y = 6.823 0.0132~ (R3 = 0.98, P
Lymphocyte blastogenesis experiments were conducted for unfractionated and fractionated ULP samples representing nonpregnant and pregnant cows. For fractionated ULP samples, five major fractions representing S-200 peaks were tested for immunosuppressive activity. Procedures for bovine lymphocyte collection and extraction techniques and the in vitro lymphocyte-PHAculture experiments have been described (11-13). Briefly, immunosuppressive activity was evaluated in sterile Falcon Micro Test III plates in which each well contained 5 x 105 bovine lymphocytes, 0.4 pg of the T-lymphocyte specific mitogen PHA (Wellcome Research Labs., Beckenham, England) in 4 ~1 of distilled water (except for one control culture),and unfractionated ULP (25 to 400 pg/ml) and S-200 ULP fractions (25 to 100 pg/ml) of varying concentrations in quadruplicate cultures. ULP was added to culture wells -10 min after the addition of lymphocytes and PHA. In a preliminary experiment, 0.4 pg of PHA yielded maximal stimulation of 5 x lo5 lymphocytes without altering their viability. Control cultures (two types) contained lymphocytes only or lymphocytes plus PHA. Cultures were incubated in humidified 95% air:5% CO2 at 37.5 C. At 48 h of culture, 0.5 PCi of 3Hthymidine (3H-3 methyl, 6.7 Ci/mmol; New England Nuclear, Boston, MA) was added to each culture well (excluding viability cultures) in 0.05 ml of complete culture medium (RPMI-1640 containing 10% fetal bovine serum, 2% extra 200 mM glutamine (v/v), 100 IU of penicillin and 100 ug of streptomycin/ml),whichyielded a total volume of 0.2 ml/culture well. Cultures were harvested with 10% trichloroaceticacid at 60 f 1 R on cellulose-triacetatefilter
JUNE 1986 VOL. 25 NO. 6
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discs using an automatic cell harvester (Skatron, Inc., Sterling, VA). Discs were placed in vials containing 8 ml of scintillation fluid and counted (8). For cultures involving the incorporation of 'H-thy&dine, parallel cultures were used to determine percentage viability of lymphocytes at the onset and termination of culture using the trypan blue exclusion method. A detailed description has been reported (9). For statistical analyses, counts per minute (cpm) for all cultures receiving 3H-thymidine were converted to picomoles (pmol) of thymidine according to a standard formula (19). To standardize immunosuppressivedata among experiments, thymidine incorporation data in test protein cultures were expressed as percentages ('$) of control (lymphocytes+ PHA) incorporation data using the following formula: 100 x (pm01 of thymidine incorporation in test protein culture-background)/(pm01 of thymidine incorporation in control culture-background). Thymidine incorporation (percent of control) and viability data were subjected to regression analysis. Individual regression lines were constructed from prediction equations in which protein concentrationwas the independent variable and percent of control and percent of viability were dependent variables. Significant R2 values reflected suppression of PHAinduced lymphocyte blastogenesis or reduced lymphocyte viability. Differences in degree of immunosuppressiveactivity at specific ULP concentrations for unfractionated ULP samples from nonpregnant and pregnant cows and among S-200 fractions within and between reproductive stages were assessed by analysis of variance considering concentration and S-200 fractions as main effects. Mean values for degree of suppression at a given concentration between pregnant and nonpregnant fractions or among S-200 fractions within a pregnancy status were separated by Duncan's multiple range test (when applicable). All analyses have been described (20). Additionally, quantitative estimates of the relative size of each fraction comprising the S-200 profiles for ULP from nonpregnant and pregnant cowswere derived by cutting out each fraction (in duplicate determinations)from the tracing paper and expressing the weight of each fraction as a percentage of the weight of the entire profile. Limited observations did not warrant a thorough statistical evaluation.
RESULTS Unfractionated Day 17 ULP from nonpregnant and pregnant beef cows suppressed (P
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nonpregnant and pregnant cows, mean percent of control values for 100 and 200 e ULP/ml were less (P~0.05) for pregnant than nonpregnant cows, thus reflecting greater immunosuppressive activity for ULP from pregnant cows. For all experiments in this investigation,mean (k SEM) pmol of thymidine for cultures containing lymphocytes alone or lymphocytes plus PHA were 1.6 f 0.2 and 24.9 + 2.2, respectively. A representative S-200 profile for fractionated ULP samples from nonpregnant and pregnant cows is illustrated (Figure 2). Five major fractions were revealed from gel filtration chromatography and the M, estimates appear above each major fraction. Protein M, estimates ranged from 5,700 to >248,000 (void volume). Therefore, fraction I may contain protein(s) of much greater Mr than 248,000. Although fractionated samples were few in number, the percentage of total protein in each fraction appeared to be similar between nonpregnant and pregnant cows, and the percentages for fractions I, II, III, IV and V were 10.7 and 7.1X, respectively 26.2 and 23.'7",respectively; 30.0 and 28.7%, respectively; 16.5 and 26.4%, respectively; and 16.7 and 14-l%, respectively.
UNFRACTIONATED PROTEIN
. . \ . % . \ .
Nonpregnant Pregnant -
SO
PROTEIN Figure 1.
100
-
150
-
\
-
260
250
360
CONCENTRATION,.ug/ml
Regression lines constructed from prediction equations illustrating the effect of bovine ULP upon PHA-stimulated lymphocyte blastogenesis. Protein secretions were collected on Day 17 of pregnancy and the estrous cycle.
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I
v I
looi
1
120
I
140
ELUTION Figure 2.
160
l60
220
200
240
VOLUME,ml
Representativegel (Sephacryl S-ZOO) filtration profile of bovine ULP collected on Day 17 of pregnancy and the estrous cycle.
Immunosuppressivedata for S-200 ULP fractions from nonpregnant cows are presented (Figure 3). All five fractions suppressed (P~O.001) lymphocyte blastogenesis to PHA. Regression equations for the individual fractions follow: I (Y = 79.3 1.039x, R2 = 0.69, P
Immunosuppressivedata for S-200 ULP fractions from pregnant cows are presented (Figure 4). All five fractions suppressed (PiO.05 to 0.001) lymphocyte blastogenesis to PHA. Regression equations for the individual fractions follow: I (y = 87.7 2.225x, R2 = 0.82, P
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PROTEIN Figure 3.
CONCENTRATION,ug/ml
Regression lines constructed from prediction equations illustrating the effect of Sephacryl S-200 fractions (I to V) of bovine ULP (Day I_7 of the estrous cycle) upon PHA-stimulated lymphocyte blastogenesis.
P
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PROTEIN Figure 4.
CONCENTRAT\ON.ug/mI
Regression lines constructed from prediction equations illustrating the effect of Sephacryl S-200 fractions (I to V) of bovine ULP (Day 17 of pregnancy) upon PHA-stimulated lymphocyte blastogenesis.
respectively (P
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FRACTION
100
I
Nonpregnan t Pregnant -
FRACTION
V
Nonpregnan t Pregnant - -
-
-
80
60
I 20
40
60
PROTEIN
Figure 5.
, 80
CONCENTRATION,
2-o
4’0
6.0
80
ug/ ml
Comparison of degree of immunosuppressiveactivity of Sephacryl S-200 ULP fractions I and V (high and low molecular weight, respectively) for cows on Day 17 of pregnancy and the estrous cycle.
DISCUSSION The degrees of immunosuppressiveactivity of Day 17 ULP secretions from nonpregnant and pregnant beef cows in this investigation were similar to the degrees of immunosuppressive activity previously reported for these preparations (11). These data further support previous data indicating that ULP secretions collected on Day 14 from ewes (8) and on Days 17 to 18 from beef cows (11-13) are more immunosuppressivein pregnant than in nonpregnant ewes and beef cows. Data in this study demonstrate that the majority of immunosuppressiveactivity resulted from at least two proteins, one of relatively low M, (-14,000) and the other considered to be of high M, (>219,000). These findings have not been previously reported for beef cattle. The low M, protein is similar in M, to the principal immunosuppressiveprotein (-15,000) previously reported for swine (6) and rabbits (21). At this time it is unknown whether more than one immunosuppressiveprotein is present in S-200 fractions I and V.
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Greater immunosuppressiveactivity of bovine ULP secretions for pregnant than nonpregnant cows, as demonstrated in this and other studies (U-13), is likely due to greater quantities, activation or numbers of immunosuppressiveproteins in S-200 fractions I (high M,) and V (low M,). Although percentage composition of fractions I and V was not greater for pregnant than nonpregnant cows, perhaps the immunosuppressiveproteins constituted a minimal percentage of total protein within each fraction. Further, although mean quantities of total ULP were identical between pregnant and nonpregnant cows (18.9and 18.9 mg, respectively) on Day 17, polypeptides of 9,000 and 15,000 Mr were quantitatively greater in ULP secretions of pregnant than nonpregnant cows (17). In another study (22), three high M, polypeptides (range of 306,800 to 342,800 M,) were present in uterine flushings on Day 19 only for pregnant cows. In this study, there were no differences in percent of control values between corresponding fractions II, III and IV for nonpregnant and pregnant cows, thus supporting the postulation that greater immunosuppressiveactivity of ULP for pregnant than nonpregnant cows results from low (14,000 and high (>219,000) Mr proteins. The low and high M, immunosuppressiveproteins identified in this investigation appear to be derived primarily from endometrial tissue; however, the conceptus may also be responsible for part of the production of the immunosuppressiveproteins. It is conceivable that the presence of the conceptus could either directly or indirectly influence the synthesis or secretion or both of the low and high M, immunosuppressiveproteins, since immunosuppressive activity of these proteins was greater in ULP secretions from pregnant than nonpregnant cows. Viability data indicate that immunosuppressiveactivity of ULP was probably not mediated by overall lymphocyte cytotoxicity, although one must consider that the viability of a specific subpopulation(s) of T-lymphocytes responsible for the suppressor activity in ULP cultures may have declined while another subpopulation(s) proliferated. Viability data are analogous to viability data previously reported for immunosuppressivestudies in beef cattle (ll-13), swine (6) and sheep (8,9). Further, previous mixed lymphocyte culture (MLC) data (11) indicate that immunosuppressionof the ULP preparations used in this investigation was not mediated by inhibition of binding of PHA to the lymphocytes. MLC data in that study (11) indicated that ULP secretions were more suppressive for pregnant than nonpregnant cows, similar to immunosuppressiveactivities observed for experiments utilizing PHA. At the termination of lymphocyte cultures containing PHA and ULP in this investigation, aggregations of lymphocytes were similar to those in control samples with PHA alone. If PHA were unable to interact with lymphocyte binding sites, aggregation of lymphocytes would seem unlikely.
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1.
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2.
Black, W. G., Simon, J., Kidder, H. E. and Wiltbank, J. N. Bactericidal activity of the uterus in the rabbit and the cow. Am. J. Vet. Res. -15:247-251 (1954).
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Hawk, H. W., Turner, G. D. and Sykes, J. F. Variation in the inflammatory and bactericidal activity of the sheep J. Vet. Res. 22: uterus during the estrous cycle. Am. 689-692 (1961).
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Reimers, T. J. and Dziuk, P. J. The survival of intrauterine skin autografts and allografts in sheep. J. Reprod. Fertil. -38:465-467 (1974).
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Segerson, E. C. and Libby, D. W. Mitogenic response of lymphocytes collected from jugular and uterine veins and the uterine lumen of estrous, day 14 and ovariectomized ewes. Biol. Reprod. -30:126-133 (1984).
6.
Murray, F. A., Segerson, E. C. and Brown, F. T. Suppression of lymphocytes in vitro by porcine uterine secretory protein. Biol. Reprod. -19:15-25 (1978).
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Murray, F. A., Zurcher, V. and Grifo, A. P. Suppression of lymphocyte reactivity in vitro by porcine allantoic and amniotic fluids. Theriogenology-11:217-225 (1979).
8.
Segerson, E. C. Immunosuppressiveeffect of ovine uterine secretory protein upon lymphocytes in vitro. Biol. Reprod. -25377-84 (1981).
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Segerson, E. C., Moffatt, R. J., Bazer, F. W. and Roberts, R. M. Suppression phytohemagglutinin-stimulatedlymphocyte blastogenesis by ovine uterine milk protein. Biol. Reprod. -30:1175-1186 (1984).
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Staples, L. D., Brown, D., Binns, R. M. and Heap, R. B. The influence of protein hormones and conceptus extracts on sheep lymphocyte transformation induced in vitro. Placenta 4:125131 (1983).
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Segerson, E. C., Libby, D. W., Getz, W. R. and Randel, R. D. Immunosuppressiveeffect of uterine secretory protein from Angus and Brahman cows upon lymphocytes in vitro. J. Anim. Sci. 2:1047-1059 (1984).
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Fisher, S. J., Gimenez, T. and Henricks, D. M. Immunosuppressive activity associated with early pregnancy in the bovine. Biol. Reprod. -32:894-906 (1985).
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Roberts, G. P. Inhibition of lymphocyte stimulation by bovine uterine proteins. J. Reprod. Fertil. -50:337-339 (1977).
14.
Murray, F. A., Bazer, F. W., Wallace, H. D. and Warnick, A. C. Quantitative and qualitative variation in the secretion of protein by the porcine uterus during the estrous cycle. Biol. Reprod. 13314-320 (1972).
15.
Squire, G. D., Bazer, F. W. and Murray, F. A. Electrophoretie patterns of porcine uterine protein secretions during the estrous cycle. Biol. Reprod. 1:321-325 (1972).
16.
Staples, L. D. Isolation and partial characterizationof an antigen associated with pregnancy in the ewe. Biol. Reprod. -22:675-685 (1980).
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Segerson, E. C., Hansen, T. R., Libby, D. W., Randel, R. D. and Getz, W. R. Ovarian and uterine morphology and function in Angus and Brahman cows. J. Anim. Sci. -59:1026-1046 (1984).
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Lowry, 0. H., Rosebrough, N. J,, Farr, A. L. and Randall, R. J_ Protein measurement with the Folin phenol reagent. J. Biol. Chem 193:265-275 (1951).
19.
Burford-Mason, A. P. and Gyte, G. M. L. An alternative method of expressing results of lymphocyte transformation experiments. J. Immunol. Methods 28:391-394 (1979).
20.
Snedecor, G. W. and Cochran, W. G. Statistical Methods. Iowa State University Press, Ames, 1967, pp. 59-62, 135-171 and 274.
21.
Mukherjee, A. B., Ulane, R. E. and Agrawal, A. K. Role of uteroglobin and transglutaminasein masking the antigenicity of implanting rabbit embryos. Am. J. Reprod. Immunol.2:135141 (1982).
22.
Bartol, F. F., Thatcher, W. W., Bazer, F. W., Kimball, F. A., Chenault, J. R., Wilcox, C. J. and Roberts, R. M. Effects of the estrous cycle and early pregnancy on bovine uterine, luteal and follicular responses. Biol. Reprod. 25:759-776. (1981).
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