The oestrous cycles of 20 mixed-breed mares were synchronized with daily injections of ..... Mitchell, G.W., Jr, Jacobs, A.A., Haddad, V., Paul, B.B.,. Strauss, R.
Bactericidal activity of peripheral blood neutrophils during the oestrous cycle and early pregnancy in the mare P. J. Strzemienski, R. M.
Dyer, R. M.
Section
P. L. Sertich, M. C. Garcia, and
Kenney
of Reproduction and tMedicine, New Bolton Center, University of Pennsylvania, School of Veterinary Medicine, Kennett Square, PA 19348, U.S.A. *
Summary. The oestrous cycles of 20 mixed-breed mares were synchronized with daily injections of 10 mg oestradiol-17\g=b\and 150 mg progesterone given i.m. for 10 days. On the 10th day, 10\p=n-\15mg prostaglandin F-2\g=a\ was administered i.m. to induce oestrus. Neutrophils were isolated from jugular blood on the 2nd or 3rd day of oestrus, Days 5 and 7 after ovulation or during early pregnancy (Days 18\p=n-\34of pregnancy). Neutrophils were challenged with Staphylococcus aureus and their bactericidal activity examined after 30 and 120 min of incubation for a reduction of colony forming units. Bactericidal activity increased with the time of incubation (P < 0\m=.\01)but did not differ for the oestrous cycle or pregnancy (P > 0\m=.\05). Introdution
During coitus, semen is deposited into the mare uterus (Millar, 1952). At this time, micro¬ organisms orginating from the penis and vagina can be transported with the semen into the uterus. Therefore the mare uterus must simultaneously control invading microflora, while supplying an environment supportive to spermatozoa. The regulatory mechanisms involved remain obscure. Spermatozoa as well as microorganisms should be recognized as 'foreign' to the uterus, but our present understanding of uterine immune response does not readily explain this apparent divergency of function. The neutrophil plays a crucial role in clearing uterine bacterial inflammation (Black et al, 1954; Hawk, 1958) and its activity can be modified by the uterus. For example, oestrogen acting antagonistically to progesterone facilitates the removal of a uterine bacterial inoculum by cervical dilatation (Black et al, 1954; Winter et al, 1960), increased uterine contractility (Black et al, 1953; Winter et al, 1960) and an increased influx of leucocytes into the uterine lumen (Brinsfield et al, 1963; Hawk, 1958; Heap et al, 1962). However, prolonged exposure of mice to oestrogens leads to pyometra (Weinstein et al, 1936; Gardner & Allen, 1937). Information on the cyclic effects of gonadal hormones on neutrophil bactericidal activity is not readily available. This information is needed for evaluating the complex nature of uterine immune mechanisms. In this study, mare peripheral blood neutrophils were challenged with Staphylococcus aureus and a common source of opsonin to determine their bactericidal function. Materials and Methods Animals. Twenty mixed-breed mares, selected as embryo transfer recipients, were used in this study. They were fed daily hay and grain ration and received all vaccinations at least 6 months before experimentation. The mares were synchronized with intramuscular injections of 150 mg progesterone and 10 mg oestradiol-17ß (Sigma Chem. Co., St Louis, MO) daily for 10 days (Loy et al, 1981). On the tenth day, 10-15 mg prostaglandin F-2a (Lutalyse; Upjohn Co., Kalamazoo, MI) was administered i.m. to lyse any remaining corpora lutea. Mares were teased daily with a stallion for signs of oestrus. Follicle size and ovulation was determined by daily palpation per rectum and ultrasonography. a
Pregnancy was established by transcervical transfer of collection of peripheral blood neutrophils.
an
embryo and
was
confirmed by ultrasonography before
Neutrophil isolation. Blood (50 ml) was collected from the jugular vein and mixed with 6 ml 3-6% (w/v) sodium citrate. Blood was collected on the 2nd or 3rd day of oestrus, the 5th and 7th day after ovulation and on Days 18, 28, 33 and 34 of pregnancy. Neutrophils were isolated as already described (Strzemienski et al, 1984) with the following modifications. The neutrophil-red blood cell pellet recovered after centrifugation over Histopaque (Sigma), was + resuspended in 2 ml Ca2 and Mg2 +-free Dulbecco's phosphate-buffered saline (pH 7-3). Sterile distilled water (6 ml) was used (20 sec) to lyse the red blood cells. Immediately, 3 ml of a 2 concentration of Dulbecco's phosphate-buffered saline containing 20mM-Hepes (pH 7-3; 0-32m) was added to restore tonicity. An additional 20ml Dulbecco's phosphate-buffered saline containing 0-22% glucose (w/v) and lOmM-Hepes (pH 7-3) (BPS) were then added. The suspension was centrifuged at room temperature (21-24°C) for 7 min at 100 g. The remaining pellet consisting of neutrophils and a small percentage of eosinophils was resuspended to a concentration of 2-94 IO6 neutrophils/ml in PBS. Neutrophil viability, assessed by exclusion of trypan blue, remained between 96 and 98% live. -
Bacterial preparation. Staphylococcus aureus (ATCC 27217) was grown in Brain Heart Infusion medium (BHI) overnight. A 6-h subculture was diluted 1:2 with 30% glycerol in Medium BHI (v/v) and frozen in aliquants ( —70°C). In preparation for the assay, an aliquant of S. aureus was thawed and added to 50 ml Medium BHI. The suspension was grown as a stationary culture for about 19 h (37°C). The top 20 ml of the S. aureus suspension were washed twice with gel water (01% gelatin in distilled water) for 10 min at 1000 g. The final pellet was adjusted to an absorbance of 01 at 620 nm for an approximate concentration of 4-5 107 colony forming units (CFU)/ml. Bactericidal assay. Based on the initial studies of Maaloe (1946), the following bactericidal assay was used. Duplicate, sterile capped 12x75 mm polystyrene test tubes were used for neutrophils of each animal. Heat-inactivated serum (56°C for 30 min) from a gelding served as the opsonin and was frozen in aliquants ( —70°C). Opsonin (150 pi) was added to 850 pi of the neutrophil suspension. Then 25 pi bacterial suspension were added and mixed. A 10 pi sample from each tube was placed in the appropriate dilution of gel water (0 time). The assay tubes were then capped, and rotated (12 rev./min) at 37°C. Tubes were resampled by removing a 10 pi sample after 30 and 120 min of incu¬ bation. Diluted samples were vortexed for 10 sec and 25 pi of the diluted sample were spread on agar plates (10% sheep blood agar) with a sterile glass rod until dry. Dilution for samples were adjusted to contain 50-300 CFU/plate. Duplicate lawn plates were prepared for each sample. The plates were then incubated in air for 24 h at 37°C and the CFU were counted. Preliminary experiments in which the opsonin had been heated at 80°C for 30 min indicated that killing of S. aureus was due to phagocytosis. Therefore, control tubes for each assay contained buffer, opsonin and
bacteria.
Progesterone determinations. Plasma progesterone determinations were performed as described by Ginther et al (1985) from jugular blood. Statistical analyses. CFU were converted to percentage bacteria killed before transformation to log10 units (Hoffman & Bullock, 1973). The transformed data were analysed by an unweighted-means analysis with 4 levels of treatment, 2 levels of time and mares within treatment (Winer, 1975). The percentage of bacteria killed is reported using the geometric mean and 95% confidence limits (Sokal & Rohlf, 1981). Student's t test was used when appropriate. Results
time, the opsonin control contained 9-2 IO5 + 2-8 IO5 (mean + s.d.) CFU/ml and did IO5 CFU/ml). differ ( > 005) from assay tubes containing neutrophils (9-4 IO5 + 3-1 Bactericidal activity after 30 min of incubation, was not different from neutrophils taken at oestrus, dioestrus and pregnancy (P > 005) (Fig. 1). Killing of S. aureus increased after 120 min of incubation (P < 001), but was not different (P > 0-05) for cycling and pregnant mares. The mean IO5 ± 2 CFU remaining after incubation with neutrophils for all treatment groups was 4-4 IO5 CFU/ml and was different (P < 001) from zero time values (9-4 IO5 ± 31 IO5 CFU/ml. Plasma progesterone concentrations for the mares are shown in Table 1. Progesterone concen¬ trations at oestrus were lower than at dioestrus (P < 001), suggesting no active corpora lutea and no residual progesterone from the synchronization regimen. At zero not
Discussion no difference in mare neutrophil antistaphylococcal activity during the oestrous cycle or early pregnancy, suggesting that oestradiol and progesterone have no lasting influence on mare neutrophil bactericidal activity. It is generally accepted that the neutrophil in vivo approaches an inflammatory site through chemotactic stimuli before initiating phagocytic and bactericidal
Our results show
100·
ría 80-
20
|5|7| |
5 7
120
30
Minutes
Fig. 1. Neutrophil bactericidal activity during the oestrous cycle and early pregnancy. Bacteria killed are reported as the geometric mean with 95% confidence intervals for oestrus ( ; 6), 6) and 7 (7; 3) after ovulation and Days 18-34 of pregnancy ( ; Days 5 (5; 4). =
=
Table 1.
=
=
Jugular plasma progesterone concentrations used as a source of neutrophils
of
mares
After ovulation Oestrus
Day 5
Day 7
Pregnancy*
7
6
3
4
15-3 ± 3-9
13-4 + 3-5
8-4 + 3-5
No. of mares
Progesterone (ng/ml)t
0-6
±0-2
»Days 18-34. tMean + s.d.
Although the rate of ingestion and killing in vivo is not known (Verhoef & Waldvogel, the results from the present study should be helpful in ascribing roles to different blood 1985), elements in affecting mare neutrophil bactericidal activity. Antistaphylococcal response was more variable at 30 min of incubation than at 120 min. This may be due to the time needed to orchestrate the intracellular killing process (Klebanoff, 1975) or may be due to the size of bacterial challenge (Clawson & Repine, 1976; Leijh et al, 1980). A contribution to bactericidal activity may also be due to neutrophil-secreted extracellular products (Verhoef & Waldvogel, 1985). Extracellular events leading to the reduction of CFU are difficult to establish, but heat treatment (56°C for 30 min) of the opsonin should minimize extracellular anti¬ staphylococcal activity due to complement. Heating the opsonin at 80°C for 30 min in preliminary events.
experiments stopped neutrophil bactericidal activity, suggesting that the bactericidal activity in these studies is intracellular. Giemsa-stained slides after 120 min of treatment also indicated bacterial
uptake by neutrophils. Progesterone values during oestrus were similar to those of previous reports (Smith et al, 1970; Vandeplassche et al, 1979), indicating no residual progesterone from the synchronization regimen or active luteal tissue. Progesterone concentrations at Days 5 and 7 after ovulation and during pregnancy are also similar to reported values (Squires et al, 1974; Holtan et al, 1975; Vandeplassche et al, 1979), suggesting a functional corpus luteum. Neutrophil bactericidal function in women also does not appear to change with pregnancy (Mitchell et al, 1970), but the utilization of autologous serum prevents the effective separation of serum influence on neutrophil bactericidal activity. In this study, the use of an almost homogenous preparation of neutrophils (some eosinophils present) should remove any direct influence from blood elements. In this way, the inherent bactericidal activity of the neutrophil is studied. Administration of oestradiol-17ß to mice reduces the bactericidal activity of peritoneal neutrophils without affecting phagocytosis (Kita et al, 1985). Low levels of myeloperoxidase in these neutrophils may account for the reduced intracellular killing (Kita et al, 1985). Phagocytic activity of the reticulo-endothelial system can be increased by synthetic oestrogens in mice (Nicol et al, 1964), but oestrogen also decreases host resistance of mice by the inhibition of interleukin-2 production (Pung et al, 1985). Preinoculation chemiluminescence response of horse neutrophils to S. zooepidemicus was not different for control and steroid-treated mares (Washburn et al, 1982; Ganjam et al, 1982) and supports the findings in this study. At 2 days after an intrauterine inoculation of S. zooepidemicus, however, chemiluminescence of peripheral neutrophils is higher in ovariectomized mares treated with oestrogen than in ovariectomized mares given progesterone (Washburn et al, 1982; Ganjam et al, 1982). Continued treatment with the hormones results in greater chemiluminescence by periph¬ eral neutrophils from progesterone-treated mares than from oestrogen-treated mares (Washburn et al, 1982). Since uterine isolates of S. zooepidemicus could be obtained only from ovariectomized mares treated with progesterone, the increased chemiluminescence of peripheral neutrophils from these mares may be due to a bacterial and/or uterine modulation of the neutrophils. In reducing the ability of the uterus to eliminate the bacteria, progesterone may indirectly influence neutrophil function by allowing the increased production of bacterial by-products. Bacteria can alter phagocyte function (Schwab, 1983) and their presence in the uterus may explain the increased chemiluminescence response of peripheral neutrophils to opsonized zymosan after prolonged progesterone treatment (Washburn et al, 1982). We thank Thornbrook Farm and A. W. Berry for his support; G. Dreisbach, M. Cahoon and L. Miller for care of the animals; and P. Brewer for secretarial assistance. Supported in part by a grant from the America Quarter Horse Association.
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Received 30 September 1986
