Journal of Reproductive Immunology,
17 (1990) 2740
27
Elsevier Scientific Publishers Ireland Ltd. JR1 00636
Lymphocyte subpopulations in lymph and blood draining from the uterus and ovary in sheep R.G. Alders* and J.N. Shelton Developmental Physiology Group, John Curtin School Universiry Canberra, ACT 2601 (Australia)
of Medical Research,
Australian National
(Accepted for publication 12 October 1989)
Summary Lymphocyte subsets in utero-ovarian peripheral lymph and uterine and jugular venous blood were analysed with the aid of monoclonal antibodies, polyclonal antisera and flow microfluorometry. The proportion of various lymphocyte subpopulations, as determined by monoclonal antibodies (mAbs) and polyclonal antisera, was found to vary between utero-ovarian peripheral lymph and jugular and uterine venous blood. T cell levels were higher in utero-ovarian peripheral lymph (approx. 80% CDS+, 50% CD4+ and 23% CD8+) than peripheral blood (approx. 55% CD5+, 18% CD4+ and 12% CDS+). Conversely, in lymph, 10% of lymphocytes were B cells compared to 30% in blood. There were 20-30% MHC II+ cells in utero-ovarian peripheral lymph and 40-50% in blood. The level of CD45R+ cells in utero-ovarian peripheral lymph was low (2%) compared to peripheral blood (approx. 55% in pregnant and 25% in non-pregnant ewes). The proportion of lymphocyte subpopulations in lymph was similar for pregnant and non-pregnant ewes. However, some differences in levels in peripheral blood were evident between uterine and jugular venous blood and pregnant and non-pregnant ewes. CD4+ cells were higher in the uterine vein (14%) than in the jugular vein (11%) of pregnant ewes. The uterine and jugular veins in pregnant ewes contained approx. 50% MHC II+ cells compared to 30% in non-pregnant ewes. Likewise, the proportion of CD45R+ cells was higher in uterine and jugular venous blood of pregnant ewes (approx. 58%) compared to non-pregnant ewes (around 25%).
*Present address: School of Veterinary Zambia.
Medicine University
of Zambia, PO Box 32379, Lusaka,
0165-0378/90/$03.50 0 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
28
Key words: lymphocyte subsets; pregnancy;
utero-ovarian lymph; sheep.
Introduction It is now widely accepted that lymphocyte distribution within the body is non-random (Butcher 1986; Miyasaka and Trnka, 1986). Lymphocytes have been found to migrate preferentially to peripheral lymph nodes, intestinal lymph nodes or sites of chronic inflammation (Smith et al. 1970; Scollay et al. 1976). Immunoregulation during pregnancy may require enhancement or diminution of the proportion of certain subpopulations of lymphocytes. In certain strains of mice, specific and non-specific suppressor cells have been found in the para-aortic lymph nodes of pregnant animals (Clark et al. 1984; Streilein and Wegmann, 1987); it is suggested that these suppressor cells play a role in the immunoregulation of pregnancy. The present work was undertaken to investigate the distribution of lymphocyte subpopulations to utero-ovarian peripheral lymph, uterine venous blood and jugular venous blood in pregnant and non-pregnant ewes using monoclonal antibodies.
Materials and Methods
Cell preparation
Samples were obtained from mature pregnant (6-120 days of gestation) and non-pregnant (4-17 days of the oestrous cycle) Merino ewes. Peripheral lymph cells were collected by cannulation of utero-ovarian lymphatics. The cannulae were constructed from clear vinyl tubing (Dural Plastics and Engineering, Dural, NSW) and were designed to ensure the mixing of lymph with heparinized Dulbecco’s phosphate buffered saline (PBS) close to the site of cannulation. Peripheral blood lymphocytes were derived from whole jugular or uterine venous blood collected in 2.7% ethylenediaminetetra-acetic acid (EDTA) disodium salt (Ajax Chemicals, Auburn NSW). The buffy coat was aspirated following centrifugation at 840 x g for 25 min. The lymphocytes were enriched by either the Ficoll-Hypaque method or by erythrocyte lysis method (Alders and Shelton 1988a). Each cell population was washed twice in PBS and resuspended at a concentration of 1 x lo6 cells/ml in Hanks’ balanced salt solution containing 5% fetal calf serum (FCS).
29
Monoclonal antibodies
Monoclonal antibodies were obtained from three different sources: The Base1 Institute for Immunology, Basel; The Sheep Biology Unit, The University of Melbourne; and The Monoclonal Antibody Center, Washington State University. The mAbs were allocated to one of two series, based on their time of arrival. Series 1. The following mAbs were obtained from The Base1 Institute for Immunology: ST-l (CD5; Beya et al., 1986); ST-8 (CD8; Ezaki et al., 1987); T-80 (T cell subset with unknown function; Ezaki et a1.,1985); and 197 (T cell subset with unknown function, T80- and ST8-; Miyasaka et al., 1985). The mAb TH81A (MHC II; Davis et al., 1987) was obtained from Washington State University. Series 2. The following mAbs were obtained from the Sheep Biology Unit: SBU-I (MHC I; Gogolin-Ewens et al., 1985); SBU-II (MHC II; Puri et al., 1985); SBU-LCA (Maddox et al., 1985a); SBUp220 (CD45R; Mackay et al., 1987); SBU-Tl (CD5; Mackay et al., 1985); SBU-T4 (CD4; Maddox et al., 1985b); SBU-T6 (CDl; Mackay et al., 1985); SBU-T8 (CD8; Maddox et al., 1985b); SBU-T19 (T cell subset which is CD4CD8- and absent from B cells; Mackay et al., 1986); and SBU3 (recognises a population of binucleate cells in trophoblast and syncytial layer in placentome; Lee et al., 1985). The quantity of lymphocytes available from utero-ovarian peripheral lymph was not sufficient to permit all the mAbs to be used in one experiment. Consequently, lymphocytes from jugular blood were used in one experiment where a comparison was made between labelling of PBL with mAbs from two sources. Polyclonal antibodies
B-cells were identified with fluorescein-conjugated rabbit anti-sheep IgG (heavy and light chains) F(ab’), fragments (Cappel Scientific Division, Cooper Biomedical Inc., Malvern, USA). Flow microjluorometry
Approximately 1 x lo6 cells were incubated with 100 yl of optimally diluted mAb at 4°C for 20 min. The optimal dilution of mAb was the greatest dilution which, when incubated with samples of cells, gave a clear separation of positive and negative cell populations. Control tubes contained an irrelevant mAb of the same subclass as the panel of mAbs employed. The cells were then incubated with 100 ~1 of a 1 : 50 dilution of affinity-purified fluorescein-conjugated sheep antimouse IgG antibody (Silenus Laboratories, Dandenon-g, Vie) at 4°C for 20 min. After each
30
incubation the cell suspension 300 x g for 5 minutes
was underlaid
with FCS, centrifuged
at
Results
Comparison of lymphocyte subpoputations, labelled by Series I mAbs, in utero-ovarian peripheral lymph and jugular venous blood of pregnant and non-pregnant ewes
A significant difference in the relative proportions of some lymphocyte subpopulations was found between jugular venous blood and utero-ovarian peripheral lymph (Table 1; Fig. 1). The mAbs ST-l, ST-8 and T-80 labelled a significantly higher proportion of cells in peripheral lymph than in jugular venous blood in both pregnant and non-pregnant ewes. Conversely, significantly less cells were labelled by antiserum to sheep IgG in peripheral lymph than in jugular venous blood, the mean values being 10.2% and 34.7% respectively, for pregnant ewes and 9.7% and 31.5% for nonpregnant ewes. Approximately twice as many lymphocytes were labelled by TH81A in jugular venous blood (40-50%) than in utero-ovarian peripheral lymph (20-30%) in both pregnant and non-pregnant ewes. There was no significant difference between the percentage of cells labelled by mAb 197 in peripheral lymph and peripheral blood. No apparent differences in lymphocyte subpopulations were found within the oestrous cycle (data not shown) or between pregnant and non-pregnant ewes (Table 1). Similarly, no significant differences were found between nulliparous or multiparous non-pregnant ewes (data not shown). During the conduct of this experiment, it became evident that peripheral blood lymphocyte (PBL) subpopulations may vary according to the technique employed to isolate the PBL (Alders and Shelton 1988a).
31
Jugular blood Peripheral lymph Jugular blood Peripheral lympth
Pregnant
54.9 k 3.7(5)d 82.8 2 2.7(8)*** 53.8 -r-2.8(7) 80.0~ 3.5(7)***
ST-l CD5 9.8 2 1.6(6) 23.0 k 2.7(g)*** 7.4 k 0.6(7) 24.6 k 2.7(7)**
ST-8 CD8
Percentage of labelled cells (kS.E.M.)
34.5 + 78.8? 47.3 f 84.3 f
2.0(2) 1.3(2)*** 3.6(4) 1.4(4)*
T-80 T cell subset 6.6 4.3 6.5 6.3
+ 1.3(2) 2 0.2(2) k 4.0(2) k 0.6(2)
197 T cell subset T80-,ST8-
labelled by Series I mAbs in jugular venous blood and utero-ovarian
*P < 0.05 for ewes of same reproductive status; **P < 0.01; ***P x 0.001. “PBL separation achieved with the use of Ficoll-Hypaque. bUnless specified there were no significant differences between compartments at P < 0.05. ‘Anti-sheep IgG(H + L), F(ab’)z fragment, Cappel Laboratories, West Chester, USA. dNumber of samples.
Non-pregnant
Source of cells
Reproductive status
Comparison of lymphocyte subpopulations pregnant ewe@.
TABLE 1
41.9 f 19.4 k 53.6 2 33.3 k
LO(2) 4.2(3) 6.6(4) 7.0(4)*
TH8lA MHC II
34.7 + 10.5(2) 10.2 + 2.1(5)* 31.5 2 5.0(4) 9.7 ” 3.1(5)*
sIg’
peripheral lymph from pregnant and non-
w
33
Comparison of lymphocyte subpoputations, labelled by Series H mAbs, in utero-ovarian peripheral lymph of pregnant and non-pregnant ewes Identification of lymphocyte subpopulations in utero-ovarian peripheral lymph with Series II mAbs revealed no significant differences (at the 5% level) between pregnant and non-pregnant ewes (Table 2). Differences were apparent, however, when the present results from utero-ovarian peripheral lymph were compared with those reported for popliteal peripheral lymph (Mackay et al. 1988). The percentage of cells labelled by the mAb SBU-T4 was higher in the utero-ovarian peripheral lymph, of pregnant (54.7%) and non-pregnant ewes (47.2%) than in popliteal lymph (38.5%). Similarly, a higher percentage of cells in utero-ovarian peripheral lymph (23.0% and 23.3%) were labelled by the mAb SBU-T8 compared to popliteal peripheral lymph (12.8%). Conversely, the mAb SBU-T19 labelled a lower proportion of cells in utero-ovarian peripheral lymph (8.0% and 4.5%) than in popliteal lymph (27.8%). A mAb (SBU-3; Lee et al. 1985), reported to label a population of binucleate cells in the trophoblast and syncytial layer in the placentome, was applied to utero-ovarian peripheral lymph cells from pregnant ewes on six occasions; no labelling of cells was detected. Comparison of lymphocyte subpoputations, labelled by Series II mAbs, in uterine and jugular venous blood of pregnant and non-pregnant ewes Lymphocytes which have migrated through tissues may leave an organ by way of the blood stream as well as by the lymphatic network (Gorman and Halliwell, 1988). This experiment was undertaken to discern if the relative composition of PBL from the uterine vein varied from that found in the jugular vein. PBL were obtained by erythrocyte lysis. There was no significant difference between uterine or jugular venous blood in the percentage of cells labelled by SBU-T1 in pregnant or nonpregnant ewes (Table 3). Likewise, no significant differences were evident in the levels of cells identified by SBU-T8 (Table 3). However, significant differences were evident in the level of expression of antigens recognized by SBU-T4, SBU-II and SBU-p220. The percentage of cells recognized by SBU-T4 was consistently higher in the uterine vein in pregnant and nonpregnant ewes (Table 3). However, the difference was not significant at the 5% level (paired t-test) and no significant difference was apparent between pregnant and non-pregnant sheep (Student's t-test). The degree of expression of MHC class II, as recognized by mAb SBUII, did not vary significantly between the jugular and uterine veins (Table 3) in pregnant or non-pregnant ewes. Interestingly though, the degree of expression was higher in pregnant ewes compared to non-pregnant ewes but the difference was significant (P < 0.05) only for samples obtained from the jugular vein. Similarly, the percentage of lymphocytes labelled by SBU-
2.8 f 0.6(4) 0.5 + 0.0(2) ND ND
44.6 -t 11.5(4) 37.3 + 4.4(3) ND 35.3
8.0? 3.0(2) 4.5 f 0.6(2) ND 27.8
0.4 2 0.1(3) 0.8 2 0.6(2) O(4) ND
23.0? 1.6(7) 23.3 f 2.9(4) ND 12.8
54.7 2 5.3(7) 47.2 & 8.4(4) 50.2 2 3.6(4) 38.5
78.8 + 6.7(4)” 78.4 * 7.0(3) NR
89.8
Pregnant Non-pregnant Hein et al., 1987
Mackay et al., 1988
“Number of samples. ND, not done.
SBU-P220 CD45R
SBU-II MHC II
SBU-T19 T cell subset CD4- ,CD8-
SBU-T6 CD1
ewes (first two
SBU-T8 CD8
lymph of pregnant and non-pregnant
SBU-T4 CD4
Percentage of labelled cells (Z..E.M.)
pheripheral
SBU-Tl CD5
Reproductive status and Previous studies
Comparison of lymphocyte subpopulations labelled by Series II mAbs in utero-ovarian rows) and popliteal peripheral lymph (Hein et al., 1987; Mackey et al., 1988).
TABLE 2
Uterine 13.9 k3.8 25.4 27.5
Jugular
60.2 23.4 55.5 27.8
Uterine
59.3 24.3 55.2 26.0
SBU-T4
SBU-Tl
Percentage of labelled cells (Y%E.M.)
10.9 A3.4 18.0 k4.6
Jugular 13.2 k1.9 14.1 20.9
Uterine
SBU-T8
13.1 22.5 14.2 22.1
Jugular
“Significantly different at P < 0.05 from other values in the column with the same superscript. bSignificantly different at P < 0.02 from other values in the column with the same superscript. bSignificantly different at P < 0.02 from other values in the column with the same superscript.
Non-pregnant
Pregnant
Reproductive status
Jugular 59.7’ k8.1 26.9’ 26.1
SBU-P220 Uterine 57.4b k8.8 20.6b k5.5
Jugular 54.0” lr8.9 25.8” rt3.0
SBU-II Uterine 53.1 28.9 32.5 k4.2
Comparison of lymphocyte subpopulations labelled by Series II mAbs in jugular and uterine venous blood of four pregnant (6-110 days of gestation) and four non-pregnant (days 6-15 of the oestrous cycle) ewes.
TABLE 3
36
~220 was significantly higher in both jugular and uterine veins in pregnant ewes compared to non-pregnant ewes (P < 0.02). The levels were similar in jugular and uterine veins for ewes of the same reproductive status. Comparison of labelling of PBL by mAbs reported to recognize the same cell surface antigen
Within the panel of mAbs used, some mAbs have been reported to recognize the same cell subsets or cell surface antigens. PBLs from two to six ewes were labelled with each of the mAbs and then those mAbs reputed to identify the same marker were statistically compared using a paired ttest. ST-l (Beya et al., 1986) and SBU-Tl (Mackay et al., 1985) have been reported to be pan T cell markers and in the current study no significant difference (P < 0.6) was found in the number of peripheral blood T lymphocytes labelled by these two mAbs (Table 4; Fig. 2). Similarly, there was no significant difference (P> 0.3) in the percentage of cells labelled by THSlA (Davis et al., 1987) or SBU-II (Puri et al., 1987), which have been reported to recognize MHC class II (Table 4). A significant difference (P < 0.02) was found, however, when PBL were labelled with ST-8 (Ezaki et al., 1987) or SBU-T8 (Maddox et al., 1985b). A consistently higher proportion of cells was labelled by SBU-T8 in comparison to that labelled by ST-8 (Table 4). Discussion The varying levels of lymphocyte subsets between utero-ovarian peripheral lymph and peripheral blood support the assertion that the exit of TABLE 4 Comparison of lymphocyte labelling by mAbs reported to recognise the same cell subsets of cell surface antigens. PBL from two to six ewes were tested for each mAb. Ewe number
Percentage of cells labelled ST-1 CD5
SBU-Tl CD5
ST-8 CD8
SBU-T8 CD8
TH81A MHC II
SBU-II MHC II
51.3 65.1 41.3 77.5 78.1 73.3
54.0 64.8 46.4 76.2 78.7 71.0
ND 6.2” 17.0 3.0 37.5 17.6
ND 12.8” 24.9 19.1 43.4 19.7
57.8 57.8 ND ND ND ND
55.7 56.7 ND ND ND ND
“Each value in the column was significantly different at P < 0.02 from the corresponding column with the same superscript. ND, not done.
value in the
31 TH81A
Fhresoenoe htendty
(IOQ)
Fig. 2. Flow cytometric analysis of PBL labelled with mAbs reported to recognize the same cell surface antigen. Monocytes were excluded from analysis by gating on forward and 90” light scatter.
lymphocyte subpopulations from the blood is non-random (Butcher, 1986; Miyasaka and Tmka, 1986; Mackay et al., 1988). The current findings and those reported by previous workers have shown that peripheral lymph contains a lower proportion of surface Ig+lymphocytes (Scollay et al., 1976; Miller and Adams, 1977; Hein et al., 1987; Mackay et al., 1988) and a higher proportion of T cells than blood or efferent lymph (Hein et al., 1987; Mackay et al., 1988). It has been suggested that the differences in the distribution of lymphocyte subpopulations between various compartments may result from lymphocyte subsets bearing different migrationrelated cell surface molecules (Butcher, 1986; Mackay et al., 1988; Streeter et al., 1988; Wu et al., 1988). Accordingly, lymphocyte migration would be directed by expression of lymphocyte surface receptors for organ specific endothelial cell determinants, which have been termed “addressins” (Streeter et al., 1988). Although proportions of cell phenotypes in utero-ovarian peripheral lymph were generally similar to those found in other peripheral lymph, there were some notable exceptions. The proportion of SBU-T8+ cells in both pregnant and non-pregnant ewes was obviously higher in utero-ovarian peripheral lymph and conversely the proportion of SBU-T19+ cells was lower in utero-ovarian peripheral lymph when compared to popliteal peripheral lymph (Mackay et al., 1988). The significance of the increased percentage -of cytotoxic lymphocytes (CD8+) and/or cytotoxic lymphocyte
38
precursors in utero-ovarian lymph is This increase be associated the immunoregulation pregnancy allevels were significantly different pregnant and ewes. In the low of SBU-p220+ in utero-ovarian lymph, despite reported high in uterine thelium (Lee al. 1988; et al., and the of India to be from the endometrium to draining lymph (Alders and 1988b), suggest uterine peripheral phatics have input from uterine endometrium. isolated from venous blood not necessarily ative of found in venous blood. one phenotypic was apparent the lymphocyte found in and jugular blood. The of SBU-T4’ in the vein was higher than in the vein. To the composition PBL subsets peripheral blood only been for samples from the vein. It be of to compare present finding values obtained PBL found blood vessels ing other regions. A report (Miyasaka McCullagh, 1981), on mixed cultivation against or third PBL and with Con or LPS, that cells from the uterus in uterine vein functionally similar those isolated the jugular As PBL separated over density gradient prior to conduct of functional assays, biological of this remains uncertain. increased number PBLs recognized the mAbs and SBU-~220 pregnant ewes interesting: the significance of difference requires investigation. In case, approx. as many were labelled pregnant ewes compared to ewes. No variable, apart the reproductive distinguished ewes. This difference in of PBL pregnant and ewes stands contrast to similarity of phenotypes found utero-ovarian peripheral of ewes various reproductive It may that in the inner of the are drained blood vasculature whereas in mouse fetal can escape the blood also the lymphatics (Streilein Wegmann, 1987). the mAb was found label a percentage of as the SBU-II, the of PBLs the jugular labelled by was not different between and non-pregnant However, a comparison between two mAbs not possible PBLs labelled TH81A were by the method and labelled with by erythrocyte Comparative studies that the ST-1 and
39
TH81A and SBU-II, reported to recognize the sheep homologues of CD5 and MHC class II respectively, label a similar proportion of PBLs. This suggested that they recognize the same cell surface antigens. However, the mAbs ST-8 and SBU-T8 labelled significantly different proportions of PBLs, suggesting that they recognize different antigens and/or cell types. A strict comparison between these two mAbs was difficult as they are of different isotypes, ST-8 being IgM and SBU-T8 being IgG, even though the control mAbs were of matching isotypes. It is noteworthy that although the elimination of ST-F cells has been shown to result in the abrogation of the in vitro generation of CTL and their effector function (Ezaki et al., 1987), no functional role of cells expressing SBU-T8 antigen has been reported. The above findings confirm the suitability of sheep for the investigation of lymphocyte migration within specific regions. Our understanding of the immunology of pregnancy is far from complete but these results have revealed areas where further investigation may prove fruitful. References Alders, R.G. and Shelton, J.N. (1988a) Analysis of ovine peripheral blood lymphocyte subsets following Ficoll-Hypaque separation or erythrocyte lysis. Res. Vet. Sci. 45, 253-254. Alders, R.G. and Shelton, J.N. (1988b) Ovine uterine lymphatics: in vivo passage of India ink from uterine subserosa, myometrium and lumen. Proc. 20th Ann. Conf. Aust. Sot. Reprod. Biol., Newcastle. p. 57, Abstr. Beya, M.F., Miyasaka, M., Dudler, L., Ezaki, T. and Tmka, Z. (1986) Studies on the differentiation of T lymphocytes in sheep. II. Two monoclonal antibodies that recognize all ovine T-lymphocytes. Immunology 57, 115-121. Butcher, E. C. (1986) The regulation of lymphocyte traffic. Curr. Top. Microbial. Immunol. 128, 85-122. Clark, D.A., Slapsys, R.M., Croy, B.A. and Rossant, J. (1984) Immunoregulation of host-versusgraft responses in the uterus. Immunol. Today 5, 111-115. Davis, W.C., Marusic, S., Lewin, H.A., Splitter, G.A., Perryman, L.E., McGuire, T.C. and Gorham J.R. (1987) The development and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the Major Histocompatibility Complex for use in the study of the immune system in cattle and other species. Vet. Immunol. Immunopathol. 15, 337-376. Ezaki, T., Miyasaka, M., Beya, M.F., Dudler, L. and Tmka, Z. (1987) A murine anti-sheep T8 monoclonal antibody, ST-8, that defines the cytotoxic T lymphocyte population. Int. Archs. Allergy Appl. Immunol. 82, 168-177. Ezaki, T., Parisot, R., Dudler, L., Beya, M.F., Miyasaka, M. and Trnka, Z. (1985) Monoclonal antibodies to surface markers which define functional subsets of T lymphocytes. In: Immunology of the Sheep (Morris B. and Miyasaka M., eds.), pp. 88-110. F. Hoffman-La Roche and Co., Basle, Switzerland. Gogolin-Ewens, K.J., Mackay, CR., Mercer, W.R. and Brandon, M.R. (1985) Sheep lymphocyte antigens (OLA). I. Major Histocompatibility Complex Class I Molecules. Immunology 56,717-723. Gorman, N.T. and Halliwell, R.E. (1988) Introduction, In: Veferinary Clinical Zmmunology, R.E. Halliwell R.E. and Gorman, N.T., eds.), W.B. Saunders Co., Philadelphia, pp.l-18. Hein, W.R., McClure, S.J. and Miyasaka, M. (1987) Cellular composition of peripheral lymph and skin of sheep defined by monoclonal antibodies. Int. Archs. Allergy appl. Immun. 84, 241-246. Lee, C.S., Gogolin-Ewens, K. and Brandon, M.R. (1988) Identificaton of a unique lymphocyte subpopulation in the sheep uterus. Immunology 63, 157-164.
40 Lee, C.S., Gogolin-Ewens, K. and White, T.R. (1985) Studies on the distribution of binucleate cells in the placenta of the sheep with a monoclonal antibody SBU-3. J. Anat. 140, 565-576. Mackay, CR., Kimpton, W.G., Brandon, M.R. and Cahill, R.N.P. (1988) Lymphocyte subsets show marked differences in their distribution between blood and the afferent and efferent lymph of peripheral lymph nodes. J. Exp. Med. 167, 1755-1765. Mackay, CR., Maddox, J.F. and Brandon, M.R. (1987) A monoclonal antibody to the ~220 component of sheep LCA identifies B cells and a unique lymphocyte subset. Cell. Immunol. 110, 46-55. Mackay, CR., Maddox, J.F. and Brandon, M.R. (1986) Three distinct subpopulations of sheep T lymphocytes. Eur. J. Immunol. 16, 19-25. Mackay, C.R., Maddox, J.F., Gogolin-Ewens, K.J. and Brandon, M.R. (1985) Characterization of two sheep lymphocyte differentiation antigens, SBU-Tl and SBU-T6. Immunology 55, 729-737. Maddox, J.F., Mackay, CR. and Brandon, M.R. (1985a) The sheep analogue of leucocyte common antigen (LCA). Immunology 55, 347-353. Maddox, J.F., Mackay, CR. and Brandon, M.R. (1985b) Surface antigens, SBU-T4 and SBU-TB, of sheep T lymphocyte subsets defined by monoclonal antibodies. Immunology 55, 739-748. Mercer, W.R., Gogolin-Ewens, K.J., Lee, C.S. and Brandon, M.R. (1987) Study of the immune response in the pregnant ovine uterus. Proc. Aust. Sot. Reprod. Biol. 19, Abstr. Miller, H.R.P. and Adams, E.P. (1977) Reassortment of lymphocytes in lymph from normal and allografted sheep. Am. J. Pathol. 87, 59-80. Miyasaka, M., Beya, M.F., Dudler, L., Parisot, R., Ezaki, T. and Trnka, Z. (1985) Studies on lymphocyte differentiation and migration in sheep by the use of monoclonal antibodies. In: Zmmunology of the Sheep Morris B. and Miyasaka M., (eds.), F. Hoffman-La Roche and Co., Basle, Switzerland, pp. 68-87. Miyasaka, M. and McCullagh, P. (1981) Immunological responsiveness of maternal and foetal lymphocytes during normal pregnancy in the ewe. J. Reprod. Immunol. 3, 15-27. Miyasaka, M. and Trnka, Z. (1986) Lymphocyte migration and differentiation in a large-animal model: the sheep. Immunol. Rev. 91, 87-114. Pm-i, N.K., Mackay, C.R. and Brandon, M.R. (1985) Sheep lymphocyte antigens (OLA). II. Major Histocompatibility complex class II molecules. Immunology 56, 725-733. Scollay, R., Hall, J. and Orlans, E. (1976) Studies on the lymphocytes of sheep. II. Some properties of cells in various compartments of the recirculating lymphocyte pool. Eur. J. Immunol. 6, 121-125. Smith, J.B., McIntosh, G.H. and Morris, B. (1970) The migration of cells through chronically inflamed tissues. J. Pathol. 100, 21-29. Streeter, P.R., Rouse, B.T.N. and Butcher, E.C. (1988) Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes. J. Cell Biol. 107, 1853-1862. Streilein, J.W. and Wegmann, T.G. (1987) Immunologic privilege in the eye and the fetus. Immunol. Today 8, 362-366. Wu, N.W., Jalkanen, S., Streeter, P.R. and Butcher, E.C. (1988) Evolutionary conservation of tissuespecific lymphocyte-endothelial cell recognition mechanisms involved in lymphocyte homing. J. Cell Biol. 107, 1845-1851.
17 (1990) 2740
27
Elsevier Scientific Publishers Ireland Ltd. JR1 00636
Lymphocyte subpopulations in lymph and blood draining from the uterus and ovary in sheep R.G. Alders* and J.N. Shelton Developmental Physiology Group, John Curtin School Universiry Canberra, ACT 2601 (Australia)
of Medical Research,
Australian National
(Accepted for publication 12 October 1989)
Summary Lymphocyte subsets in utero-ovarian peripheral lymph and uterine and jugular venous blood were analysed with the aid of monoclonal antibodies, polyclonal antisera and flow microfluorometry. The proportion of various lymphocyte subpopulations, as determined by monoclonal antibodies (mAbs) and polyclonal antisera, was found to vary between utero-ovarian peripheral lymph and jugular and uterine venous blood. T cell levels were higher in utero-ovarian peripheral lymph (approx. 80% CDS+, 50% CD4+ and 23% CD8+) than peripheral blood (approx. 55% CD5+, 18% CD4+ and 12% CDS+). Conversely, in lymph, 10% of lymphocytes were B cells compared to 30% in blood. There were 20-30% MHC II+ cells in utero-ovarian peripheral lymph and 40-50% in blood. The level of CD45R+ cells in utero-ovarian peripheral lymph was low (2%) compared to peripheral blood (approx. 55% in pregnant and 25% in non-pregnant ewes). The proportion of lymphocyte subpopulations in lymph was similar for pregnant and non-pregnant ewes. However, some differences in levels in peripheral blood were evident between uterine and jugular venous blood and pregnant and non-pregnant ewes. CD4+ cells were higher in the uterine vein (14%) than in the jugular vein (11%) of pregnant ewes. The uterine and jugular veins in pregnant ewes contained approx. 50% MHC II+ cells compared to 30% in non-pregnant ewes. Likewise, the proportion of CD45R+ cells was higher in uterine and jugular venous blood of pregnant ewes (approx. 58%) compared to non-pregnant ewes (around 25%).
*Present address: School of Veterinary Zambia.
Medicine University
of Zambia, PO Box 32379, Lusaka,
0165-0378/90/$03.50 0 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
28
Key words: lymphocyte subsets; pregnancy;
utero-ovarian lymph; sheep.
Introduction It is now widely accepted that lymphocyte distribution within the body is non-random (Butcher 1986; Miyasaka and Trnka, 1986). Lymphocytes have been found to migrate preferentially to peripheral lymph nodes, intestinal lymph nodes or sites of chronic inflammation (Smith et al. 1970; Scollay et al. 1976). Immunoregulation during pregnancy may require enhancement or diminution of the proportion of certain subpopulations of lymphocytes. In certain strains of mice, specific and non-specific suppressor cells have been found in the para-aortic lymph nodes of pregnant animals (Clark et al. 1984; Streilein and Wegmann, 1987); it is suggested that these suppressor cells play a role in the immunoregulation of pregnancy. The present work was undertaken to investigate the distribution of lymphocyte subpopulations to utero-ovarian peripheral lymph, uterine venous blood and jugular venous blood in pregnant and non-pregnant ewes using monoclonal antibodies.
Materials and Methods
Cell preparation
Samples were obtained from mature pregnant (6-120 days of gestation) and non-pregnant (4-17 days of the oestrous cycle) Merino ewes. Peripheral lymph cells were collected by cannulation of utero-ovarian lymphatics. The cannulae were constructed from clear vinyl tubing (Dural Plastics and Engineering, Dural, NSW) and were designed to ensure the mixing of lymph with heparinized Dulbecco’s phosphate buffered saline (PBS) close to the site of cannulation. Peripheral blood lymphocytes were derived from whole jugular or uterine venous blood collected in 2.7% ethylenediaminetetra-acetic acid (EDTA) disodium salt (Ajax Chemicals, Auburn NSW). The buffy coat was aspirated following centrifugation at 840 x g for 25 min. The lymphocytes were enriched by either the Ficoll-Hypaque method or by erythrocyte lysis method (Alders and Shelton 1988a). Each cell population was washed twice in PBS and resuspended at a concentration of 1 x lo6 cells/ml in Hanks’ balanced salt solution containing 5% fetal calf serum (FCS).
29
Monoclonal antibodies
Monoclonal antibodies were obtained from three different sources: The Base1 Institute for Immunology, Basel; The Sheep Biology Unit, The University of Melbourne; and The Monoclonal Antibody Center, Washington State University. The mAbs were allocated to one of two series, based on their time of arrival. Series 1. The following mAbs were obtained from The Base1 Institute for Immunology: ST-l (CD5; Beya et al., 1986); ST-8 (CD8; Ezaki et al., 1987); T-80 (T cell subset with unknown function; Ezaki et a1.,1985); and 197 (T cell subset with unknown function, T80- and ST8-; Miyasaka et al., 1985). The mAb TH81A (MHC II; Davis et al., 1987) was obtained from Washington State University. Series 2. The following mAbs were obtained from the Sheep Biology Unit: SBU-I (MHC I; Gogolin-Ewens et al., 1985); SBU-II (MHC II; Puri et al., 1985); SBU-LCA (Maddox et al., 1985a); SBUp220 (CD45R; Mackay et al., 1987); SBU-Tl (CD5; Mackay et al., 1985); SBU-T4 (CD4; Maddox et al., 1985b); SBU-T6 (CDl; Mackay et al., 1985); SBU-T8 (CD8; Maddox et al., 1985b); SBU-T19 (T cell subset which is CD4CD8- and absent from B cells; Mackay et al., 1986); and SBU3 (recognises a population of binucleate cells in trophoblast and syncytial layer in placentome; Lee et al., 1985). The quantity of lymphocytes available from utero-ovarian peripheral lymph was not sufficient to permit all the mAbs to be used in one experiment. Consequently, lymphocytes from jugular blood were used in one experiment where a comparison was made between labelling of PBL with mAbs from two sources. Polyclonal antibodies
B-cells were identified with fluorescein-conjugated rabbit anti-sheep IgG (heavy and light chains) F(ab’), fragments (Cappel Scientific Division, Cooper Biomedical Inc., Malvern, USA). Flow microjluorometry
Approximately 1 x lo6 cells were incubated with 100 yl of optimally diluted mAb at 4°C for 20 min. The optimal dilution of mAb was the greatest dilution which, when incubated with samples of cells, gave a clear separation of positive and negative cell populations. Control tubes contained an irrelevant mAb of the same subclass as the panel of mAbs employed. The cells were then incubated with 100 ~1 of a 1 : 50 dilution of affinity-purified fluorescein-conjugated sheep antimouse IgG antibody (Silenus Laboratories, Dandenon-g, Vie) at 4°C for 20 min. After each
30
incubation the cell suspension 300 x g for 5 minutes
was underlaid
with FCS, centrifuged
at
Results
Comparison of lymphocyte subpoputations, labelled by Series I mAbs, in utero-ovarian peripheral lymph and jugular venous blood of pregnant and non-pregnant ewes
A significant difference in the relative proportions of some lymphocyte subpopulations was found between jugular venous blood and utero-ovarian peripheral lymph (Table 1; Fig. 1). The mAbs ST-l, ST-8 and T-80 labelled a significantly higher proportion of cells in peripheral lymph than in jugular venous blood in both pregnant and non-pregnant ewes. Conversely, significantly less cells were labelled by antiserum to sheep IgG in peripheral lymph than in jugular venous blood, the mean values being 10.2% and 34.7% respectively, for pregnant ewes and 9.7% and 31.5% for nonpregnant ewes. Approximately twice as many lymphocytes were labelled by TH81A in jugular venous blood (40-50%) than in utero-ovarian peripheral lymph (20-30%) in both pregnant and non-pregnant ewes. There was no significant difference between the percentage of cells labelled by mAb 197 in peripheral lymph and peripheral blood. No apparent differences in lymphocyte subpopulations were found within the oestrous cycle (data not shown) or between pregnant and non-pregnant ewes (Table 1). Similarly, no significant differences were found between nulliparous or multiparous non-pregnant ewes (data not shown). During the conduct of this experiment, it became evident that peripheral blood lymphocyte (PBL) subpopulations may vary according to the technique employed to isolate the PBL (Alders and Shelton 1988a).
31
Jugular blood Peripheral lymph Jugular blood Peripheral lympth
Pregnant
54.9 k 3.7(5)d 82.8 2 2.7(8)*** 53.8 -r-2.8(7) 80.0~ 3.5(7)***
ST-l CD5 9.8 2 1.6(6) 23.0 k 2.7(g)*** 7.4 k 0.6(7) 24.6 k 2.7(7)**
ST-8 CD8
Percentage of labelled cells (kS.E.M.)
34.5 + 78.8? 47.3 f 84.3 f
2.0(2) 1.3(2)*** 3.6(4) 1.4(4)*
T-80 T cell subset 6.6 4.3 6.5 6.3
+ 1.3(2) 2 0.2(2) k 4.0(2) k 0.6(2)
197 T cell subset T80-,ST8-
labelled by Series I mAbs in jugular venous blood and utero-ovarian
*P < 0.05 for ewes of same reproductive status; **P < 0.01; ***P x 0.001. “PBL separation achieved with the use of Ficoll-Hypaque. bUnless specified there were no significant differences between compartments at P < 0.05. ‘Anti-sheep IgG(H + L), F(ab’)z fragment, Cappel Laboratories, West Chester, USA. dNumber of samples.
Non-pregnant
Source of cells
Reproductive status
Comparison of lymphocyte subpopulations pregnant ewe@.
TABLE 1
41.9 f 19.4 k 53.6 2 33.3 k
LO(2) 4.2(3) 6.6(4) 7.0(4)*
TH8lA MHC II
34.7 + 10.5(2) 10.2 + 2.1(5)* 31.5 2 5.0(4) 9.7 ” 3.1(5)*
sIg’
peripheral lymph from pregnant and non-
w
33
Comparison of lymphocyte subpoputations, labelled by Series H mAbs, in utero-ovarian peripheral lymph of pregnant and non-pregnant ewes Identification of lymphocyte subpopulations in utero-ovarian peripheral lymph with Series II mAbs revealed no significant differences (at the 5% level) between pregnant and non-pregnant ewes (Table 2). Differences were apparent, however, when the present results from utero-ovarian peripheral lymph were compared with those reported for popliteal peripheral lymph (Mackay et al. 1988). The percentage of cells labelled by the mAb SBU-T4 was higher in the utero-ovarian peripheral lymph, of pregnant (54.7%) and non-pregnant ewes (47.2%) than in popliteal lymph (38.5%). Similarly, a higher percentage of cells in utero-ovarian peripheral lymph (23.0% and 23.3%) were labelled by the mAb SBU-T8 compared to popliteal peripheral lymph (12.8%). Conversely, the mAb SBU-T19 labelled a lower proportion of cells in utero-ovarian peripheral lymph (8.0% and 4.5%) than in popliteal lymph (27.8%). A mAb (SBU-3; Lee et al. 1985), reported to label a population of binucleate cells in the trophoblast and syncytial layer in the placentome, was applied to utero-ovarian peripheral lymph cells from pregnant ewes on six occasions; no labelling of cells was detected. Comparison of lymphocyte subpoputations, labelled by Series II mAbs, in uterine and jugular venous blood of pregnant and non-pregnant ewes Lymphocytes which have migrated through tissues may leave an organ by way of the blood stream as well as by the lymphatic network (Gorman and Halliwell, 1988). This experiment was undertaken to discern if the relative composition of PBL from the uterine vein varied from that found in the jugular vein. PBL were obtained by erythrocyte lysis. There was no significant difference between uterine or jugular venous blood in the percentage of cells labelled by SBU-T1 in pregnant or nonpregnant ewes (Table 3). Likewise, no significant differences were evident in the levels of cells identified by SBU-T8 (Table 3). However, significant differences were evident in the level of expression of antigens recognized by SBU-T4, SBU-II and SBU-p220. The percentage of cells recognized by SBU-T4 was consistently higher in the uterine vein in pregnant and nonpregnant ewes (Table 3). However, the difference was not significant at the 5% level (paired t-test) and no significant difference was apparent between pregnant and non-pregnant sheep (Student's t-test). The degree of expression of MHC class II, as recognized by mAb SBUII, did not vary significantly between the jugular and uterine veins (Table 3) in pregnant or non-pregnant ewes. Interestingly though, the degree of expression was higher in pregnant ewes compared to non-pregnant ewes but the difference was significant (P < 0.05) only for samples obtained from the jugular vein. Similarly, the percentage of lymphocytes labelled by SBU-
2.8 f 0.6(4) 0.5 + 0.0(2) ND ND
44.6 -t 11.5(4) 37.3 + 4.4(3) ND 35.3
8.0? 3.0(2) 4.5 f 0.6(2) ND 27.8
0.4 2 0.1(3) 0.8 2 0.6(2) O(4) ND
23.0? 1.6(7) 23.3 f 2.9(4) ND 12.8
54.7 2 5.3(7) 47.2 & 8.4(4) 50.2 2 3.6(4) 38.5
78.8 + 6.7(4)” 78.4 * 7.0(3) NR
89.8
Pregnant Non-pregnant Hein et al., 1987
Mackay et al., 1988
“Number of samples. ND, not done.
SBU-P220 CD45R
SBU-II MHC II
SBU-T19 T cell subset CD4- ,CD8-
SBU-T6 CD1
ewes (first two
SBU-T8 CD8
lymph of pregnant and non-pregnant
SBU-T4 CD4
Percentage of labelled cells (Z..E.M.)
pheripheral
SBU-Tl CD5
Reproductive status and Previous studies
Comparison of lymphocyte subpopulations labelled by Series II mAbs in utero-ovarian rows) and popliteal peripheral lymph (Hein et al., 1987; Mackey et al., 1988).
TABLE 2
Uterine 13.9 k3.8 25.4 27.5
Jugular
60.2 23.4 55.5 27.8
Uterine
59.3 24.3 55.2 26.0
SBU-T4
SBU-Tl
Percentage of labelled cells (Y%E.M.)
10.9 A3.4 18.0 k4.6
Jugular 13.2 k1.9 14.1 20.9
Uterine
SBU-T8
13.1 22.5 14.2 22.1
Jugular
“Significantly different at P < 0.05 from other values in the column with the same superscript. bSignificantly different at P < 0.02 from other values in the column with the same superscript. bSignificantly different at P < 0.02 from other values in the column with the same superscript.
Non-pregnant
Pregnant
Reproductive status
Jugular 59.7’ k8.1 26.9’ 26.1
SBU-P220 Uterine 57.4b k8.8 20.6b k5.5
Jugular 54.0” lr8.9 25.8” rt3.0
SBU-II Uterine 53.1 28.9 32.5 k4.2
Comparison of lymphocyte subpopulations labelled by Series II mAbs in jugular and uterine venous blood of four pregnant (6-110 days of gestation) and four non-pregnant (days 6-15 of the oestrous cycle) ewes.
TABLE 3
36
~220 was significantly higher in both jugular and uterine veins in pregnant ewes compared to non-pregnant ewes (P < 0.02). The levels were similar in jugular and uterine veins for ewes of the same reproductive status. Comparison of labelling of PBL by mAbs reported to recognize the same cell surface antigen
Within the panel of mAbs used, some mAbs have been reported to recognize the same cell subsets or cell surface antigens. PBLs from two to six ewes were labelled with each of the mAbs and then those mAbs reputed to identify the same marker were statistically compared using a paired ttest. ST-l (Beya et al., 1986) and SBU-Tl (Mackay et al., 1985) have been reported to be pan T cell markers and in the current study no significant difference (P < 0.6) was found in the number of peripheral blood T lymphocytes labelled by these two mAbs (Table 4; Fig. 2). Similarly, there was no significant difference (P> 0.3) in the percentage of cells labelled by THSlA (Davis et al., 1987) or SBU-II (Puri et al., 1987), which have been reported to recognize MHC class II (Table 4). A significant difference (P < 0.02) was found, however, when PBL were labelled with ST-8 (Ezaki et al., 1987) or SBU-T8 (Maddox et al., 1985b). A consistently higher proportion of cells was labelled by SBU-T8 in comparison to that labelled by ST-8 (Table 4). Discussion The varying levels of lymphocyte subsets between utero-ovarian peripheral lymph and peripheral blood support the assertion that the exit of TABLE 4 Comparison of lymphocyte labelling by mAbs reported to recognise the same cell subsets of cell surface antigens. PBL from two to six ewes were tested for each mAb. Ewe number
Percentage of cells labelled ST-1 CD5
SBU-Tl CD5
ST-8 CD8
SBU-T8 CD8
TH81A MHC II
SBU-II MHC II
51.3 65.1 41.3 77.5 78.1 73.3
54.0 64.8 46.4 76.2 78.7 71.0
ND 6.2” 17.0 3.0 37.5 17.6
ND 12.8” 24.9 19.1 43.4 19.7
57.8 57.8 ND ND ND ND
55.7 56.7 ND ND ND ND
“Each value in the column was significantly different at P < 0.02 from the corresponding column with the same superscript. ND, not done.
value in the
31 TH81A
Fhresoenoe htendty
(IOQ)
Fig. 2. Flow cytometric analysis of PBL labelled with mAbs reported to recognize the same cell surface antigen. Monocytes were excluded from analysis by gating on forward and 90” light scatter.
lymphocyte subpopulations from the blood is non-random (Butcher, 1986; Miyasaka and Tmka, 1986; Mackay et al., 1988). The current findings and those reported by previous workers have shown that peripheral lymph contains a lower proportion of surface Ig+lymphocytes (Scollay et al., 1976; Miller and Adams, 1977; Hein et al., 1987; Mackay et al., 1988) and a higher proportion of T cells than blood or efferent lymph (Hein et al., 1987; Mackay et al., 1988). It has been suggested that the differences in the distribution of lymphocyte subpopulations between various compartments may result from lymphocyte subsets bearing different migrationrelated cell surface molecules (Butcher, 1986; Mackay et al., 1988; Streeter et al., 1988; Wu et al., 1988). Accordingly, lymphocyte migration would be directed by expression of lymphocyte surface receptors for organ specific endothelial cell determinants, which have been termed “addressins” (Streeter et al., 1988). Although proportions of cell phenotypes in utero-ovarian peripheral lymph were generally similar to those found in other peripheral lymph, there were some notable exceptions. The proportion of SBU-T8+ cells in both pregnant and non-pregnant ewes was obviously higher in utero-ovarian peripheral lymph and conversely the proportion of SBU-T19+ cells was lower in utero-ovarian peripheral lymph when compared to popliteal peripheral lymph (Mackay et al., 1988). The significance of the increased percentage -of cytotoxic lymphocytes (CD8+) and/or cytotoxic lymphocyte
38
precursors in utero-ovarian lymph is This increase be associated the immunoregulation pregnancy allevels were significantly different pregnant and ewes. In the low of SBU-p220+ in utero-ovarian lymph, despite reported high in uterine thelium (Lee al. 1988; et al., and the of India to be from the endometrium to draining lymph (Alders and 1988b), suggest uterine peripheral phatics have input from uterine endometrium. isolated from venous blood not necessarily ative of found in venous blood. one phenotypic was apparent the lymphocyte found in and jugular blood. The of SBU-T4’ in the vein was higher than in the vein. To the composition PBL subsets peripheral blood only been for samples from the vein. It be of to compare present finding values obtained PBL found blood vessels ing other regions. A report (Miyasaka McCullagh, 1981), on mixed cultivation against or third PBL and with Con or LPS, that cells from the uterus in uterine vein functionally similar those isolated the jugular As PBL separated over density gradient prior to conduct of functional assays, biological of this remains uncertain. increased number PBLs recognized the mAbs and SBU-~220 pregnant ewes interesting: the significance of difference requires investigation. In case, approx. as many were labelled pregnant ewes compared to ewes. No variable, apart the reproductive distinguished ewes. This difference in of PBL pregnant and ewes stands contrast to similarity of phenotypes found utero-ovarian peripheral of ewes various reproductive It may that in the inner of the are drained blood vasculature whereas in mouse fetal can escape the blood also the lymphatics (Streilein Wegmann, 1987). the mAb was found label a percentage of as the SBU-II, the of PBLs the jugular labelled by was not different between and non-pregnant However, a comparison between two mAbs not possible PBLs labelled TH81A were by the method and labelled with by erythrocyte Comparative studies that the ST-1 and
39
TH81A and SBU-II, reported to recognize the sheep homologues of CD5 and MHC class II respectively, label a similar proportion of PBLs. This suggested that they recognize the same cell surface antigens. However, the mAbs ST-8 and SBU-T8 labelled significantly different proportions of PBLs, suggesting that they recognize different antigens and/or cell types. A strict comparison between these two mAbs was difficult as they are of different isotypes, ST-8 being IgM and SBU-T8 being IgG, even though the control mAbs were of matching isotypes. It is noteworthy that although the elimination of ST-F cells has been shown to result in the abrogation of the in vitro generation of CTL and their effector function (Ezaki et al., 1987), no functional role of cells expressing SBU-T8 antigen has been reported. The above findings confirm the suitability of sheep for the investigation of lymphocyte migration within specific regions. Our understanding of the immunology of pregnancy is far from complete but these results have revealed areas where further investigation may prove fruitful. References Alders, R.G. and Shelton, J.N. (1988a) Analysis of ovine peripheral blood lymphocyte subsets following Ficoll-Hypaque separation or erythrocyte lysis. Res. Vet. Sci. 45, 253-254. Alders, R.G. and Shelton, J.N. (1988b) Ovine uterine lymphatics: in vivo passage of India ink from uterine subserosa, myometrium and lumen. Proc. 20th Ann. Conf. Aust. Sot. Reprod. Biol., Newcastle. p. 57, Abstr. Beya, M.F., Miyasaka, M., Dudler, L., Ezaki, T. and Tmka, Z. (1986) Studies on the differentiation of T lymphocytes in sheep. II. Two monoclonal antibodies that recognize all ovine T-lymphocytes. Immunology 57, 115-121. Butcher, E. C. (1986) The regulation of lymphocyte traffic. Curr. Top. Microbial. Immunol. 128, 85-122. Clark, D.A., Slapsys, R.M., Croy, B.A. and Rossant, J. (1984) Immunoregulation of host-versusgraft responses in the uterus. Immunol. Today 5, 111-115. Davis, W.C., Marusic, S., Lewin, H.A., Splitter, G.A., Perryman, L.E., McGuire, T.C. and Gorham J.R. (1987) The development and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the Major Histocompatibility Complex for use in the study of the immune system in cattle and other species. Vet. Immunol. Immunopathol. 15, 337-376. Ezaki, T., Miyasaka, M., Beya, M.F., Dudler, L. and Tmka, Z. (1987) A murine anti-sheep T8 monoclonal antibody, ST-8, that defines the cytotoxic T lymphocyte population. Int. Archs. Allergy Appl. Immunol. 82, 168-177. Ezaki, T., Parisot, R., Dudler, L., Beya, M.F., Miyasaka, M. and Trnka, Z. (1985) Monoclonal antibodies to surface markers which define functional subsets of T lymphocytes. In: Immunology of the Sheep (Morris B. and Miyasaka M., eds.), pp. 88-110. F. Hoffman-La Roche and Co., Basle, Switzerland. Gogolin-Ewens, K.J., Mackay, CR., Mercer, W.R. and Brandon, M.R. (1985) Sheep lymphocyte antigens (OLA). I. Major Histocompatibility Complex Class I Molecules. Immunology 56,717-723. Gorman, N.T. and Halliwell, R.E. (1988) Introduction, In: Veferinary Clinical Zmmunology, R.E. Halliwell R.E. and Gorman, N.T., eds.), W.B. Saunders Co., Philadelphia, pp.l-18. Hein, W.R., McClure, S.J. and Miyasaka, M. (1987) Cellular composition of peripheral lymph and skin of sheep defined by monoclonal antibodies. Int. Archs. Allergy appl. Immun. 84, 241-246. Lee, C.S., Gogolin-Ewens, K. and Brandon, M.R. (1988) Identificaton of a unique lymphocyte subpopulation in the sheep uterus. Immunology 63, 157-164.
40 Lee, C.S., Gogolin-Ewens, K. and White, T.R. (1985) Studies on the distribution of binucleate cells in the placenta of the sheep with a monoclonal antibody SBU-3. J. Anat. 140, 565-576. Mackay, CR., Kimpton, W.G., Brandon, M.R. and Cahill, R.N.P. (1988) Lymphocyte subsets show marked differences in their distribution between blood and the afferent and efferent lymph of peripheral lymph nodes. J. Exp. Med. 167, 1755-1765. Mackay, CR., Maddox, J.F. and Brandon, M.R. (1987) A monoclonal antibody to the ~220 component of sheep LCA identifies B cells and a unique lymphocyte subset. Cell. Immunol. 110, 46-55. Mackay, CR., Maddox, J.F. and Brandon, M.R. (1986) Three distinct subpopulations of sheep T lymphocytes. Eur. J. Immunol. 16, 19-25. Mackay, C.R., Maddox, J.F., Gogolin-Ewens, K.J. and Brandon, M.R. (1985) Characterization of two sheep lymphocyte differentiation antigens, SBU-Tl and SBU-T6. Immunology 55, 729-737. Maddox, J.F., Mackay, CR. and Brandon, M.R. (1985a) The sheep analogue of leucocyte common antigen (LCA). Immunology 55, 347-353. Maddox, J.F., Mackay, CR. and Brandon, M.R. (1985b) Surface antigens, SBU-T4 and SBU-TB, of sheep T lymphocyte subsets defined by monoclonal antibodies. Immunology 55, 739-748. Mercer, W.R., Gogolin-Ewens, K.J., Lee, C.S. and Brandon, M.R. (1987) Study of the immune response in the pregnant ovine uterus. Proc. Aust. Sot. Reprod. Biol. 19, Abstr. Miller, H.R.P. and Adams, E.P. (1977) Reassortment of lymphocytes in lymph from normal and allografted sheep. Am. J. Pathol. 87, 59-80. Miyasaka, M., Beya, M.F., Dudler, L., Parisot, R., Ezaki, T. and Trnka, Z. (1985) Studies on lymphocyte differentiation and migration in sheep by the use of monoclonal antibodies. In: Zmmunology of the Sheep Morris B. and Miyasaka M., (eds.), F. Hoffman-La Roche and Co., Basle, Switzerland, pp. 68-87. Miyasaka, M. and McCullagh, P. (1981) Immunological responsiveness of maternal and foetal lymphocytes during normal pregnancy in the ewe. J. Reprod. Immunol. 3, 15-27. Miyasaka, M. and Trnka, Z. (1986) Lymphocyte migration and differentiation in a large-animal model: the sheep. Immunol. Rev. 91, 87-114. Pm-i, N.K., Mackay, C.R. and Brandon, M.R. (1985) Sheep lymphocyte antigens (OLA). II. Major Histocompatibility complex class II molecules. Immunology 56, 725-733. Scollay, R., Hall, J. and Orlans, E. (1976) Studies on the lymphocytes of sheep. II. Some properties of cells in various compartments of the recirculating lymphocyte pool. Eur. J. Immunol. 6, 121-125. Smith, J.B., McIntosh, G.H. and Morris, B. (1970) The migration of cells through chronically inflamed tissues. J. Pathol. 100, 21-29. Streeter, P.R., Rouse, B.T.N. and Butcher, E.C. (1988) Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes. J. Cell Biol. 107, 1853-1862. Streilein, J.W. and Wegmann, T.G. (1987) Immunologic privilege in the eye and the fetus. Immunol. Today 8, 362-366. Wu, N.W., Jalkanen, S., Streeter, P.R. and Butcher, E.C. (1988) Evolutionary conservation of tissuespecific lymphocyte-endothelial cell recognition mechanisms involved in lymphocyte homing. J. Cell Biol. 107, 1845-1851.
