Expression of major histocompatibility complex (MHC) class II antigens in the murine mammary gland

Immunology Letters, 35 (1993) 7 12 0165 2478 / 93 / $ 6.00 ~) 1993 Elsevier Science Publishers B.V. All rights reserved IMLET 01875

Expression of major histocompatibility complex (MHC) class II antigens in the murine mammary gland J.L. Fitzpatrick, A.D. Wilson, P.W. Bland a n d C.R. Stokes Department of Veterinary Medicine, University of Bristol, School of Veterinary Science, Langford, Avon, UK (Received 21 August 1992; accepted 21 September 1992)

1.

Summary

Major histocompatibility complex (MHC) class II antigens were identified on cells within mammary gland connective tissue of lactating mice using a paraformaldehyde-lysine-periodate-gluteraldehyde fixative and an immunoperoxidase staining method. The distribution of class II expressing cells within interalveolar and interlobular connective tissue was similar both throughout lactation and in successive lactations. Epithelial cells within secretory alveoli and mammary ducts did not express class II antigens. 2.

Introduction

Major histocompatibility complex (MHC) class II antigens are polymorphic, integral membrane molecules which have been described on a wide variety of cells involved in the induction and regulation of immune responses [1,2]. Cells within several different non-lymphoid tissues in various species also express class II antigens, including the epithelium of the gut [3], respiratory tract and mammary gland [4,5]. In the mammary gland of the guinea-pig, epithelial class II expression is induced by pregnancy and lactation, or by the exo-

geneous administration of lactotrophic hormones [6]. HLA-DR (class II-like) antigens have been demonstrated on lactating human breast epithelium and in the membrane of human milk-fat globules, which are assumed to be derived from the epithelial cell membrane during apocrine secretion [7]. The function of class II molecules expressed by non-lymphoid tissues remains unclear, but as epithelial cells from the gut [8,9], thyroid [10] and kidney [11] have all been shown to be capable of presenting antigen in vitro, they may play a role in induction of local immune responses. In this report we describe the distribution of class II antigens in the mammary gland tissue of lactating mice. 3. 3.1.

Correspondence to: J.L. Fitzpatrick, Dept. of Veterinary Medicine, University of Bristol, School of Veterinary Science, Langford, Avon, BS18 7DU, UK.

Animals

Mammary gland tissue was collected from conventionally reared BALB/c mice, in their first, second and third lactations, on days 1, 3, 7, 10, 13, 15 and 21 of lactation. Samples were also taken occasionally from older mice in their fifth and sixth lactations. Samples were taken from three individual mice at each time point. 3.2.

Key words: MHC class II antigen; Mammary gland; Epithelial cell

Materials and Methods

Tissues

Pieces of tissue (5 mm 3) were obtained from the right caudal mammary gland of each mouse following anaesthesia and cervical dislocation. Similarly sized pieces of tissue were removed from the

spleen and from the mid-jejunum of the gastrointestinal tract. The spleen and gut tissues were used as positive control sections against which class I1 expression in mammary gland tissues was compared, as class II expression in the murine gastrointestinal tract has previously been described using this fixation and staining method [12].

3.3. Tissue fixation Tissues were fixed by a modification of the method of Gendelman et al. [13]. Briefly, tissues were added to freshly prepared fixative containing 30 parts 0.1 M lysine-HC1 in 0.05 M phosphate buffer (pH 7.4)/7.15 parts 0.1 M phosphate buffer (pH 7.4)/1.25 parts 8% (w/v) paraformaldehyde solution/1.6 parts 25% gluteraldehyde solution, to which sodium m-periodate was added at a final concentration of 0.02 M. Following overnight fixation at 4°C, samples were processed through one change of 70% alcohol, one change of 90% alcohol, two changes of 100% alcohol and two changes of xylene, each change being 45 rain at 4°C. After wax embedding at 56°C for 20 min in a vacuum embedder, samples were stored at 4°C.

3.4. Immunoperoxidase staining Sections (4/~m) were dewaxed and then rehydrated. Sections were washed twice, for 2 rain and 10 min respectively, in Tris-buffered saline (TBS, pH 7.6) between each step of the staining method. Endogenous peroxidase was blocked by incubating sections in a freshly prepared mixture containing 5 parts methanol/4 parts PBS/one part 30% hydrogen peroxide. Tissue sections were incubated in a 0.01 mg/ml solution of protease (type XIV; Sigma, Poole, Dorset) in TBS for 10 min at 37°C. In order to reduce non-specific binding of immunoglobulins, sections were then incubated with 50 #1 of a 10% solution of normal mouse serum in TBS for 30 min at 20c'C. The monoclonal antibody rat anti-mouse class II (M5/144, directed against polymorphic determinants of I-A and I-E products of the M H C in BALB/c strain mice [14]; Hybritech, California) was diluted 1:500 and added in 50-/zl volumes directly into the normal mouse serum on the slides,

to give a final dilution of 1:1000. The slides were incubated for 2-3 h at 20°C. The slides were then incubated with affinity purified biotinylated sheep anti-rat antiserum which had been adsorbed against mouse immunoglobulins (Jackson Immunoresearch, Baltimore MD, USA), at a dilution of 1:1000 for 90 rain at room temperature. Streptavidin-biotin horseradish peroxidase complexes (Dako Ltd.) were added according to the manufacturers' recommendations. Colour was developed by a 15-min incubation at 20~C with 0.6 mg/ml diaminobenzidine tetrahydrochloride (DAB; Sigma) in 0.05 M Tris-HC1, containing 0.03% hydrogen peroxide. After counterstaining with haematoxylin, the slides were dehydrated, then mounted in DPX (Merck Ltd., Poole, Dorset). Control sections had either TBS, or normal rat serum substituted for the primary antibody stage. Sections were examined by light microscopy for the presence of class II antigens. 4.

Results

Cells expressing class II antigens were identified in the interalveolar and interlobular connective tissue of lactating mammary gland tissue of mice at all time points examined. The stained cells appeared spindle-shaped and some had long cytoplasmic processes extending between the alveoli, suggesting that they were macrophages or dendritic cells (Figs. 1 and 2). Epithelial cells in mammary ducts and alveoli did not express class II at any time point examined. Some stained cells appeared to be within the alveolar epithelium but did not have the same morphology as the secretory epithelium. These cells may be macrophages or monocytes lying between secretory cells of the epithelium or in the process of traversing the epithelium. No stained cells were seen in the lumina of alveoli or ducts in the present study. No difference in class II expression was seen between groups of mice at each time point examined or between successive lactations. Even the oldest mice showed no increase in the number of stained cells in the connective tissue and the epithelium remained unstained after five or six lactations. The mice showed no evidence of mammary

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Fig. 1. lmmunoperoxidase staining of class II antigens in murine mammary tissue: day 10 of 2nd lactation. This section shows spindle-shaped class II expressing cells in the interalveolar connective tissue (arrows). x 400.

Fig. 2. immunoperoxidase staining of class 11 antigens in murine mammary tissue: day 3 of 3rd lactation. This section shows class Ii expressing cells lying between secretory epithelial cells (arrows). x 1000.

infections and no cellular infiltrate was seen histologically. Samples from the small intestine showed the

expected distribution o f class II antigens as previously described [3,12]. Class II was detected on lamina propria histiocytes and on epithelial cells

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5.

rive tissue o f lactating mice. The m a m m a r y ductular and secretory epithelial tissue did not express class II and the distribution o f staining in the gland remained unchanged t h r o u g h o u t lactation and with successive lactations.

Discussion References

The ability o f m a m m a r y gland epithelial cells to express class II antigens varies considerably between species, b o t h during lactating and non-lactating stages. The lack o f epithelial class II expression in this murine study is in contrast to the situation in lactating guinea-pigs and h u m a n s where the secretory epithelium was seen to be strongly stained for class II [6,7]. Lee et al. [15] described the distribution o f class II expressing cells in the m a m m a r y gland o f pregnant and n o n - p r e g n a n t sheep and showed that cells expressing class II antigens were f o u n d in the connective tissue a r o u n d ducts and alveoli and in some subepithelial areas o f the m a m m a r y gland o f sheep, but not in the epithelium. H o w ever, in the non-lactating bovine m a m m a r y gland, class II expressing cells were seen in both connective tissue and epithelium, particularly in the area o f the gland cistern [16]. It therefore seems unlikely that epithelial class II expression is solely a result o f h o r m o n a l influences and that immunological mediators such as interferon-7 [17] and other cytokines m a y be involved [18]. The class II expressing cells occasionally identified within the alveolar epithelium m a y have been m a c r o p h a g e s traversing the epithelium, although double-staining methods would be required to confirm this assumption. Helminen and Ericsson [19] identified m a c r o p h a g e s within the epithelial alveoli o f involuting rat m a m m a r y glands using electron microscopy. They suggest that the function o f these cells m a y be to digest degenerated epithelial cells. In s u m m a r y , ceils expressing class II were identified in the interlobular and interalveolar connec-

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