Lymphocyte subsets in the mammary gland of sows

Research in Veterinary Science 1993, 55,351-355

Lymphocyte subsets in the mammary gland of sows N. CHABAUDIE, C. LE JAN, M. OLIVIER, H. SALMON, Laboratoire de Pathologie Porcine, XNRA 37380 Nouzilly, France

The presence and localisation of lymphocyte subsets together with class II bearing cells in the mammary gland of sows, were studied at different periods of the reproductive cycle by immunohistochemistry and compared with blood. All cell types involved in the immune response were present in the mammary gland at the different stages of gestation and lactation and nearer the alveolar epithelium as gestation proceeded: T lymphocytes, including CD4+ and CD8+, B lymphocytes and class II bearing cells (epithelial ceils and macrophages). The results indicated an early accumulation of T lymphocytes, specifically T helper cells, during pregnancy; the specific increase of IgA lymphocytes occurring after this phase could suggest a role for these T cells in the induction of IgA response. The local accumulation of immune cells sustains the view that the mammary gland is able to mount a true local immune response and the increase in CD8+ cells near the epithelium suggests a role in local immune defence. THE mammary gland undergoes mammogenesis and involution phases under hormonal control. This results in the development of its local immune system for each period of gestation and lactation followed by its regression in the resting periods. The immune functions of the mammary gland are its local defence and the protection of the neonate which is transferred to the neonate in the form of antibodies and lymphoid cells via their secretions. The modulation of mammary gland immune responses, for local defence enhancement and the improvement of lactogenic immunity, needs a better knowledge of the mammary gland lymphoid cells and of their mechanism of antigenic stimulation. The capacity for a local response by the sow's mammary gland has been established by Saif and Bohl (1979, 1983). In lactation, intramammary

inoculation of attenuated gastroenteritis virus induces a predominance of specific IgA in milk: if intramammary administration occurs during pregnancy, specific IgG predominates. Responses in the mammary gland necessitate the presence of helper T cells, and of cells expressing class II molecules for the presentation of antigen. Cytotoxic T lymphocytes can be involved in protection against viral infections such as gastroenteritis virus which has a tropism for mammary epithelial cells. The evolution of the different types of lymphocytes in the mammary gland of pregnant and lactating sows has been previously established by Salmon and Delouis (1982). Using their data the present authors have looked at different periods of the sow reproductive cycle for the presence and localisation of cells which may be involved in different processes of the immune response: the humoral response of the mammary gland (IgA+, IgM+ and IgG+ lymphocytes), the amplification of immune mechanisms (CD4+ lymphocytes), antiviral activity (CD8+ lymphocytes) and antigen presentation (cells expressing class II molecules). At the same time, the proportions of the different subsets of lymphocytes were evaluated in blood. Materials and methods

Animals Mammary tissue and blood samples were obtained from five primiparous sows which represented five periods: two periods during pregnancy (day 80 and day 105 of pregnancy), one colostral period (day 0), one lactating period (day 16) and one resting period which represented the control.

Blood samples

351

The sows were killed at the same times and

352

N. Chabaudie, C. Le Jan, M. Olivier, H. Salmon

blood was collected and mixed with heparin (10 iu ml-1). After centrifugation for 30 minutes at 700 g, the buffy coat was isolated and frozen at -70°C. Then 12 gm sections were cut on a microtome. Sections were fixed in acetone for 20 minutes, then dried, frozen quickly at -70°C and stored at -70°C until staining.

Mammary tissue samples Tissue samples were taken from the last quarters of the glands of the killed sows. Skin and fatty tissue were removed and small cubes of 1 cm 3 were cut from the mammary parenchyma. The cubes of tissue were quickly frozen in liquid nitrogen, then placed in a freezing chamber at -70°C. One cube was selected at random and sectioned at 5 gm on a microtome. The sections were treated as above.

nise the endogenous peroxidase positive cells (macrophages) in the tissue, sections were incubated with hydrogen peroxide for 30 minutes and with diaminobenzidine (DAB) in the presence of cobalt chloride for 20 minutes. Peroxidase positive cells appeared black. Then, tissues were incubated with streptavidin-biotin-peroxidase complex (1/400, Amersham Laboratory) for half an hour and with the DAB tO reveal the positive cells. Lastly, tissue sections were put into the following baths, for three minutes: osmic acid, (which enhances the coloration), tap water, Harris haematoxylin, tap water, tap water and acetic acid (2 per cent), tap water, lithium carbonate, hot tap water, alcohol 70 ° , alcohol 90 ° , alcohol 100 ° and toluene. Then the sections were mounted in resin (Eukitt) and observed with an optical microscope with interference contrast (Leitz Wetzlar x 400).

Expression of results Antibodies Mouse anti-pig T helper (PT4), mouse anti-pig T cytotoxic (PT8) from Pescovitz (Pescovitz et al 1984) mouse anti-pig T cells (MSA4) from Hammerberg (Hammerberg and Schurig 1986) and mouse anti-pig IgA (K61) from Stevens, University of Bristol are monoclonal antibodies and were used as culture supematant. Mouse class II alloserum (Cederlane) is an antimouse aI alloserum which reacts with porcine a1 antigens (Shinohara and Sachs 1982): it was used at a 1/5 dilution in phosphate buffered saline (PBS) Goat anti-pig IgG and goat anti-pig IgM (Nordic Immunology) are polyclonal antibodies and were used at a 1/5 dilution in pBs. The second antibody was an anti-mouse biotinylated antibody (Amersham Laboratory). The optimum concentration of this antiserum was determined after assays using different dilutions.

Immunoperoxidase technique Sections were washed in PBS and v(ere incubated in a humid chamber for one hour with the first antibody. Sections incubated with ELY 10 (Earle lactalbumin yeast and 10 per cent fetal calf serum) medium were used as negative controls. Thereafter, the Amersham technique was used. Briefly, sections were washed in pBs and incubated for 60 minutes with the biotinylated antibodies. Between each step, sections were washed in PBS. To recog-

For the mammary gland, 100 fields at random (a field depicts a rectangle of 0.03 ram2), for each stage studied, were observed and positive cells were counted. Results are given as the number of positive cells for 100 microscopic fields. For blood, for each section, 500 cells were observed. Results are given as the number of positive cells per 100 leucocytes. Results

Presence and localisation of the T lympleocyte subsets in the mammary gland The results show that in the mammary gland (Fig 1A) and in blood (Fig 1B), the number of T cells (as judged by the monoclonal antibody against the sheep red blood cell receptor [MSA4]) was equal to the sum of the CD4+ and CD8+ cells. In the mammary gland (Fig 1A), during the resting period, the number of positive T cells was approximately 2.5 cells per field. There was an increase, from 2.5 to five on day 80 of pregnancy. Then, the number of T cells decreased from five to three on day 105 of pregnancy and remained at this level during lactation. The CD4 and CD8 positive cells showed a similar trend with a predominance of CD8+ over CD4+ cells, except on day 105 of pregnancy. This resulted in a higher CD4/CD8 ratio on day 105 of pregnancy than on day 80, 1.05 and 0.68, respectively (Table 1). In contrast, in the corresponding period, no change

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Lymphocytes in porcine mammary glands 6

TABLE 2: E v o l u t i o n o f t h e p e r c e n t a g e

A

number of positive cells

of CD4+ and CD8+

c e l l s in c l o s e c o n t a c t w i t h t h e e p i t h e l i u m o f m a m m a r y

Resting

5

CD4+ cells CD8+ cells

3

0* 0

Day of Pregnancy 80 105 4.8 13.2

acini

Day in lactating period 1

8.9 23

30 55

2

* Results are the numbers of positive cells in close contact with the epithelium of the mammary acini per 100 positive cells in the whole mammary tissue

1 -H //

0

I Day80

I Day105

~ t - C D 4 + culls

Pregnant --$~CD8. cells

ReaUng

100

I Dsyl

I Day23

Presence and Iocalisation of the B lymphocyte subsets (Fig 2)

lactaUn9 " - e - T cells

Number of positive ceils ~

B

80

j

60

o

~

e

40 ~

0 J.

~.

-&

20 o

// Reeling

i DayS0

i Day106 Pregnlnt CD4* ceils ~ CD8* cells

k Day1

__

i Day23

lactating - e - T culls

FIG 1 : Evolution of T (O), CD4+ (A) and CD8+ (~) cells in mammary gland (A) and blood (B) of resting, pregnant and lactating sows. A Number of positive cells per unit area of mammary tissue from a count of 50 fields. B Number of positive cells per 100 leucocytes

was detected in the CD4/CD8 ratio in the blood, which remained at about 0.80 (Table 1). Studies on the localisation of CD4+ and CD8+ cells (Table 2), relative to the epithelium of the mammary acini, showed the absence of CD4+ and CD8+ cells near the epithelium of mammary acini during the resting period. In contrast, there was a progressive increase in the number of these cells in close contact with the alveolar epithelium during pregnancy and the lactating period, higher for CD8+ cells than CD4+ cells (Table 2). Later, on day 16, it was not possible to evaluate these proportions precisely because of the poor architecture of the alveolar tissue. TABLE 1: Evolution of the CD4/CD8 r a t i o in t h e m a m m a r y g l a n d a n d c o r r e s p o n d i n g b l o o d of r e s t i n g , p r e g n a n t a n d lactating s o w s Resting

Mammary gland Blood

0.56 0-87

Day of pregnancy 80 105 0-68 0.8

1-05 0.75

Day in lactating period 1 23 0.80 0.72

0.61 0.77

During the first period of pregnancy no increase in the Ig+ cells in the mammary gland was observed. From day 105 to day 16 of lactation, a regular increase of the IgA+ cells was noted, and a short increase for IgG+ and IgM+ cells from day 105 of pregnancy and day 0 of lactation, respectively. During lactation, IgA+ cells increased progressively from 1 to 2.5 cells per field and predominated over IgM+ and IgG+ ceils. These Ig+ cells seemed to be predominantly lymphoblastoid on day 105 of pregnancy and plasma cells during the lactating period (data not shown). In contrast, the number of Ig+ cells remained stable in the blood of the sows during resting, pregnant and lactating periods (data not shown).

Presence of antigens on epithelial cells aI antigen was observed on the epithelium of mammary gland (alveolar and intralobular duct), as well as on the endothelial cells. Very few positive ceils were present in the interalveolar connec2.5

number of positive cells per field ~

1J

/

i

0.5

Resting

Dayl~0

Day105 pregnant -e-- IgM+ cells ~t-- igO + cells

Day1,

Day16

lactating --.¢- IgA+ cells

FIG 2: Evolution of IgA+ (A), IgG (t) and IgM+ (0) cells in the mammary gland of resting, pregnant and lactating sows. *Number of positive cells per unit area of mammary tissue from a count of 50 fields

354

N. Chabaudie, C. Le Jan, M. Olivier, H. Salmon

tive tissue. This aI antigen was observed in resting, pregnant and lactating sows. Apparently, there was no change in the density of aI antigens on the cells (data not shown). Discussion As previously shown by one of the authors (Salmon and Delouis 1982), T lymphocytes are present in the mammary glands of pregnant and lactating sows. In addition, the presence of CD4+ and CD8+ lymphocytes has been demonstrated. CD8+ lymphocytes predominated over CD4+ lymphocytes each time they were measured in mammary glands as well as in blood, except on day 105 of pregnancy, when identical proportions of CD4+ and CD8+ lymphocytes were observed in mammary glands. The findings on blood buffy coat sections extend and confirm previous studies (Pescovitz et al 1984, Salmon 1987) which showed a predominance of CD8+ over CD4+ cells in pig blood. Thus it appears that the relative predominance of CD4+ cells in the mammary gland mimics the situation in blood except on day 105, due to a decrease of CD8+ cells. This decrease does not seem to be due to the insensitivity of the technique since it was also detected in the blood at the same time. These results indicate an early accumulation of T lymphocytes, specifically T helper cells, which suggests a role for them in the induction of the IgA response. This is in agreement with results established in rats by Parmely and Manning (1983). Lee et al (1989) have shown, in mammary glands of resting and pregnant sheep, a high number of T lymphocytes, predominantly CD8+, near duct and alveolar epithelium, which is in concordance with the present authors observations of high numbers of CD8+ lymphocytes near alveolar epithelium in mammary gland of pig. The specific increase of IgA lymphocytes in lactation which occurred after the phase of increase of T lymphocytes in the mammary gland is in accordance with the findings of Parmely and Manning (1983) in rats. Furthermore, these results extend to pigs the observation made in rodents that IgA+ cells increase after parturition (Weiss Carrington 1977, Parmely 1985). All the types of cells involved in the immune response are present in the mammary gland at the different stages of gestation and lactation: T lyre-

phocytes, including CD4+ and CD8+, B lymphocytes and cells expressing aI molecules. Class II antigens are expressed by the alveolar and duct epithelium, endothelial cells and some cells in interalveolar connective tissue, with the same intensity in the mammary glands of resting, pregnant and lactating sows. In contrast, Klareskog et al (1980) in guinea pigs and Newman (1980) in man have observed that the expression of class II antigens, absent in the resting mammary gland, appears and increases during the evolution of gestation and lactation. This discrepancy could be related to a species particularity or to the use of a cross-reactive reagent. The local accumulation of T helper lymphocytes, B lymphocytes and class II antigen-bearing cells sustains the view that the mammary gland is able to respond actively to a local antigenic stimulation Furthermore, the accumulation of CDS+ cells near the epithelium may suggest that they have a role in the local protection of the mammary gland itself. Two mechanisms may be involved in the constitution of the IgA plasmacyte pool in the mammary gland. First, there may be a migration of precursors of IgA plasmacytes, which would terminate their differentiation in plasma cells in mammary tissue. Secondly a migration of lymphoblasts in the mammary gland can be hypothesised, which would expand locally in collaboration with helper T cells in accordance with the authors' observation of lymphoblastoid cells occurring before plasma cells. A fuller comprehension of migration mechanisms in mammary gland would need a comparison of the proportions of IgA lymphocytes and IgA lymphoblasts in the mammary gland and intestine during pregnancy and lactation, and a study of cytokine secretions in the mammary gland. References HAMMERBERG, C. & SCHURIG, G. (1986) Characterization of monoclonal antibodies directed against swine leukocytes. Veterinary Immunology and Immunopathology l l , 107-121 KLARESKOG, L., FORSUM, U. & PETERSON, P. A. (1980) Hormonal regulation of the expression of la antigens on mammary gland epithelium. European Journal of Immunology 10, 958-963 LEE, C. S., MEEUSEN, E. & BRANDON, M. R. (1989) Subpopulations of lymphocytes in the mammary gland of sheep. Immunology 66, 388-393 NEWMAN, R. A. (1980). The presence of HLA-DRantigen on lactating human breast epithelium and milk fat globule membrane. Clinical and Experimental Immunology 41,478-486

Lymphocytes in porcine mammary glands PARMELY, M. J. (1985) Kinetics of mammary and intestinal IgA producing cells in the lactating rat. Journal of Reproductive Immunology 8, 89-93 PARMELY, M. I. & MANNING, L. S. (1983) Cellular determinants of mammary cell-mediated immunity in the rat: kinetics of lymphocyte subset accumulation in the rat mammary gland during pregnancy and lactation. Annals of the New York Academy of Sciences 409, 517-532 PESCOWITZ, M. D., LUNNEY, J. K. & SACHS, D. H. (1984) Preparation and characterisation of monoclonal antibodies reactive with porcine PBL. Journal of Immunology 133, 368-375 SAW, L. J. & BOHL, E. H. (1979) Role of the secretory IgA in passive immunity of swine to enteric viral infection. Immunology of Breast Milk. Ed R. L. Ogra and D. Dayton. Dublin, Raven Press pp 237-255 SAIF, L. J. & BOHL, E. H. (1983) Passive immunity to transmissible gastroenteritis virus: intramammary viral inoculation of sows. Annals of the New York Academy of Sciences 409, 708-722

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SALMON, H. (1987) The intestinal and mammary immune system in pigs. VeterinaryImmunology and lmmunopathology 17, 367-388 SALMON, H. & DELOUIS, C. (1982) Cindtique des sous population lymphocytaires darts la mamelle de truie primipare en relation avec la gestation et la lactation. Annales de Recherches Vdt6rinaires 13, 4/-49 SCHINOHARA, N. & SACHS, D. H. (1982) Interspecies cross-reactions of murine anti-IA alloantibodies. IA Antigens. Vol 1 Mice. Ed S. Ferrone and G, S. David. New, York, CRC Press. pp 219-240 WEISS CARRINGTON, B. (1977) Plasma cells and immunoglobulins in the mouse mammary gland during pregnancy and lactation. Journal of Immunology 119, 1306-1309

Received August 4, 1992 Accepted June 14, 1993