Veterinary Immunology and Immunopathology, 32 (1992) 13-23

13

Elsevier Science Publishers B.V., Amsterdam

MHC class II expression in the bovine mammary gland J.L. Fitzpatricka, P.J. Crippsa, A.W. Hillb, P.W.

Bland a and C.R. Stokes a

"Department of Veterinary Medicine, Universityof Bristol, School of Veterinary Science, Langford, UK bAFRC Institute for Animal Health, Compton Laboratory, Compton, UK (Accepted 19 June 1991 )

ABSTRACT Fitzpatrick, J.L., Cripps, P.J., Hill, A.W., Bland, P.W. and Stokes, C.R., 1992. MHC class II expression in the bovine mammary gland. Vet. Immunol. Immunopathol., 32:13-23. The distribution of major histocompatibilitycomplex (MHC) class II positive cells within the connective tissue and the epithelium of the involuted bovine mammary gland has been determined. The effect of intramammary adminstration of the antigens ovalbumin and formalin killed Streptococcus uberis on the distribution pattern has also been investigated. Infusion of formalin killed S. uberis increased cellular expression of class II antigens when compared with quarters either infused with ovalbumin, not infused at all, or from which minor pathogens had been isolated. The increased expression occurred particularly in the area of the gland cistern-secretory tissue junction.

ABBREVIATIONS MHC, major histocompatibility complex; PBS, phosphate-buffered saline; TBS, tris-buffered saline. INTRODUCTION

The application of control measures for mastitis has halved the incidence of udder disease (Bramley and Dodd, 1984); however, mastitis in cattle remains a serious economic and welfare problem. Studies on local immunisation in the cow, where the mammary gland was infused with different antigens during the dry period, demonstrated that the locally produced antibody persisted throughout the ensuing lactation (Wilson, 1972). There remains a need to understand the mechanisms responsible for the generation of immune responses in the udder if future vaccination against mastitis is to be successful. Major histocompatibility complex (MHC) class II antigens are polymorphic, integral membrane molecules which have been described on a wide © 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05.00

14

J.L. FITZPATRICK ET AL.

variety of cells involved in the induction and regulation of immune responses ( Unanue et al., 1984 ). A variety of'non-professional' immune cells have also been shown to express class II antigens (Wiman et al., 1978; Daar et al., 1984). Class II expression can be induced or modified in various tissues by lymphokines (Skoskiewicz et al., 1985 ), and by immunological stimuli (Barclay and Mason, 1982 ). Class II antigens have been identified in the mammary gland of guinea-pigs, where they have been shown to be induced by pregnancy and lactation or by the exogenous administration of lactotrophic hormones (Klareskog et al., 1980). In this study, using a monoclonal antibody and an avidin-biotin immunoperoxidase technique on paraformaldehyde-lysine-periodate-glutaraldehyde fixed tissues, the presence of class II antigens on cells within the connective tissue and the epithelium of the involuted bovine mammary gland have been identified. It was also shown that class II expression on mammary gland epithelial cells can be induced by intramammary administration of formalin killed Streptococcus uberis. MATERIALS AND METHODS

Animals Nine adult Friesian cows due to be culled from the daily herd at the Institute for Animal Health, Compton, Newbury, were used in this study. All animals were non-lactating and had been so for periods varying from 2 to 8 months. All quarters of all animals had been given antibiotic therapy after the last milking (Orbenin Dry Cow, Beecham Animal Health, Brentford, U K ) . Secretions from all four quarters of each gland were examined bacteriologically shortly before administration of the antigens. Some quarters were found to be infected with minor pathogens, primarily coagulase-negative staphylococci or Corynebacterium bovis. However, with one exception, only quarters free of bacteria had antigen introduced (Table 1 ).

Antigens Ovalbumin (50 mg or 1 g) (Grade V, Sigma, Poole, U K ) was dissolved in 10 ml sterile phosphate-buffered saline (PBS; pH 7.2 ). The solution was filtered (Minisart NML, Sartorius, Belmont, UK; 0.21 #m). Six cows received 50 mg ovalbumin, and three cows had 1 mg introduced into one quarter. Streptococcus uberis strain C197C (capsulated), was isolated from a field case of bovine mastitis. The bacteria were grown for 18 h in 10 ml of Todd Hewitt broth (Oxoid, Basingstoke, U K ) , to give approximately 109 cfu ml-1 (colony forming units). The cells were then washed twice in PBS, suspended in 1% formalin in PBS for 18 h at 4 ° C, washed four times in sterile PBS and

MHCCLASSII EXPRESSIONIN THE COW

15

TABLE 1

Proportion of 20 fields in the heavily stained group I in the epithelium and connective tissue of the gland cistern-secretory tissue junction area of the mammary gland Cow no.

3 4 2 5 1 6 9 8 7

Time 2

1h 5h 18 h 48 h 96 h 144h 1h 18 h 48 h

S. uberis quarter3

Ova quarter4

Control quarter s

Control quarter

1

2

EP 6

CT 7

EP

CT

EP

CT

EP

CT

0.3 0.0 0.45 0.8 0.7 0.3 0.0 0.0 1.0

0.3 0.05 0.0 0.0 0.3 0.5 0.0 0.0 0.45

0.0 0.0 0.2 0.0 0.0"* 0.05 0.4 t 0.1TM 0.05 t

0.05 0.15 0.05 0.0 0.0"* 0.05 OA t 0.05 TM 0.25 t

0.25 0.0" 0.05 0.0" 0.0 0.0 0.35 0.0 0.0

0.1" 0.0" 0.1" 0.0 0. l 0.05* 0.25 0.05 0.0

0.0" 0.5* 0.0 0.0 0.0 0.1" 0.4 0.0 0.0

0.1 0.65* 0.05 0.0" 0.05 0.0" 0.25 0.0 0.05

~The heavily stained group showed a heavy degree of class II-positive staining. 2Time between infusion of the antigen and point of slaughter. 3Quarters infused with approximately 10 ~° cfu ofS. uberis. 4Quarters infused with 50 mg of ovalbumin (Ova) except those marked t which were infused with 1 g of ovalbumin. SControl quarters which were not infused. 6Epithelium. 7Connective tissue. *Minor pathogens isolated from these quarters. **Ten fields only examined.

finally resuspended in 10 ml of PBS. The material was checked for sterility before 10 ml of the suspension was introduced into one quarter of each of the nine cows. After thorough cleaning of the teat end, the 10 ml vols. of ovalbumin or S. uberis were administered into separate quarters via a cannula (18-gauge), introduced through the teat canal into the teat cistern, followed by hand massage of the teat towards the secretory tissue. The remaining two quarters acted as controls. Antigens were administered 1, 5, 18, 48, 96 or 144 h prior to slaughter. Tissues Small pieces of tissue (5 m m 3), were obtained from the teat duct, teat sinus, gland cistern-secretory tissue junction and secretory tissue. Tissues were fixed by a modification of the method of Gendelman et al. (1983). 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 and 1.6 parts 25% glutar-

16

J.L. FITZPATRICK ET AL.

aldehyde 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 ofxylene, each change being 45 min long and at 4°C. After wax embedding at 56°C for 20 min in a vacuum embedder, samples were stored at 4 ° C.

Immunoperoxidase staining Tissue sections of 4/~m were de-waxed in xylene and taken through alcohol to water. Endogenous peroxidase was blocked by incubating sections in a freshly prepared mixture containing 5 parts methanol, 5 parts PBS, and 1 part 30% hydrogen peroxide, for 30 min at room temperature. After washing in 0.05 M Tris-buffered saline (TBS; pH 7.6), the slides were incubated in a 0.01 mg m l - l solution ofprotease (type XIV, Sigma, Poole, U K ) , in TBS for 10 min at 37 °C. Following washing in TBS, the slides were incubated with 50 #1 of a 10% solution of normal mouse serum in TBS for 30 min at room temperature, in order to reduce non-specific binding of immunoglobulins. The monoclonal antibody ILA-21 (anti-bovine class II (directed against an invariant region, I. Morrison, personal communication, 1990), a kind gift from ILRAD, Nairobi ), was diluted 1 : 10 000 and added in 50 #1 vols. directly into the normal mouse serum on the slides, to give a final concentration of 1 : 20 000. The slides were incubated overnight at 4 ° C. Following a TBS wash, the slides were incubated with affinity-purified biotinylated rabbit anti-mouse antiserum (Dako, High Wycombe, U K ) , at a dilution of 1 : 500 for 90 min at room temperature. Following a TBS wash, the streptavidin-biotin complexhorseradish peroxidase (Dako, High Wycombe, U K ) , was added according to the manufacturers recommendations, and incubated for 1 h at room temperature. Following a final TBS wash, the colour was developed by a 15-min incubation at room temperature with a 0.6 mg ml-1 solution of diaminobenzidine tetrahydrochloride (DAB, Sigma, Poole, U K ) , in 0.05 M Tris-HC1, containing 0.03% hydrogen peroxide. After counterstaining with haematoxylin, the slides were dehydrated then mounted in DPX (Merck, Poole, U K ) . Control sections had either PBS, or normal mouse serum substituted for the monoclonal antibody stage.

Quantitation of immunoperoxidase staining Scoring of the degree of immunoperoxidase staining in the tissue sections was then carried out in a blind fashion by the same observer on each occasion. Previous studies using other observers (J.L. Fitzpatrick unpublished observations, 1990), showed similar interpretations of the degree of staining regardless of observer. Separate scores were made for the epithelium of ducts and alveoli, and inter-lobular and inter-alveolar connective tissue areas.

MHC CLASS II EXPRESSION IN THE COW

17

Quantitation was carded out only on samples obtained from the secretory tissue and the gland cistern-secretory tissue junction areas. Sections were dichotomised into a heavily stained group or a not heavily stained group. The data were analysed by a generalised linear model using maximum likelihood estimates for a binomial distribution with a logit link, adding terms sequentially into the model in what was considered to be a biologically appropriate order. This order was: ( 1 ) cow; (2) area of gland (gland cistern-secretory tissue junction or secretory tissue); (3) tissue (epithelium of ducts and alveoli or inter-lobular and inter-alveolar connective tissue); (4) treatment, (i) infusion with S. uberis, (ii) infusion with ovalbumin, (iii) presence of minor pathogens in the quarter, or (iv) control quarter which has not been infused and in which no minor pathogens were isolated); ( 5 ) the interaction of treatment with the cow. At each sequential stage of the regression analysis, the sign and magnitude of each regression coefficient was recorded. This was done in order to see how consistent the regression coefficients were when extra terms were added to the model. RESULTS

Immunoperoxidase staining Immunoperoxidase staining was carded out on sections from various areas of the mammary tissue. Marked differences were noticed in the degree of staining between fields of the same section and between the different areas of the same quarter.

General histological observations Secretory tissue and gland cistern-secretory tissue junction In all quarters examined, class II-positive cells were seen both in the interalveolar and inter-lobular connective tissue. Many positive cells had the morphological appearance of macrophages. Positive cells were often seen in close apposition to the bilayered epithelium of ducts or to the epithelium of the alveolar remnants. A number of cells appeared to be in the process of traversing the epithelium. When compared with control quarters which had not been infused, quarters which had received antigen and those from which minor pathogens had been isolated prior to intramammary administration of the antigens showed a greater degree of positive staining in the epithelial cells lining ducts and involuting alveoli. The positive epithelial staining appeared to be particularly strong in quarters receiving S. uberis (Figs. 1 and 2). Control quarters generally showed less staining in the epithelium (Fig. 3 ).

18

J.L. FITZPATRICK ET AL.

Fig. 1. Secretory tissue area, immunoperoxidase staining of class II antigens - S. u b e r i s infused into this quarter 5 h before slaughter. Many strongly stained class II-positive cells (arrows) are seen in the connective tissue (CT). The epithelium (E) of the ducts is strongly class II-positive (arrows) in some areas and less so in others. Cells accumulated in the duct lumens (L) are also positively stained (arrows). ×40.

Fig. 2. Gland cistern-secretory tissue junction area, immunoperoxidase staining of class II antigens - S. u b e r i s infused into this quarter 96 h before slaughter. This section shows class IIpositive cells (arrows) in the connective tissue area (CT). The alveolus shows marked positive staining (arrows) of the epithelial cells (E) surrounding the alveolar lumen (L). × 100.

MHC CLASS II EXPRESSION IN THE COW

19

Fig. 3. Secretory tissue area, immunoperoxidase staining of class II antigens - control quarter into which nothing was infused and no minor pathogens were previously isolated. There are a few class II-positive cells (arrows) in the interalveolar connective tissue (CT), and some lying just below or within the alveolar/ductular epithelium (E). The epithelial cells surrounding the duct lumens (L) show no staining. × 100.

Teat sinus Positive cells were seen in the subepithelial connective tissue. The epithelial cells of the teat sinus were usually either unstained or weakly stained. Occasionally, some quarters showed intense staining o f the bilayered epithelium, especially the lumenal layer (Fig. 4).

Teat duct The stratified squamous epithelium o f the teat duct was not stained. Positive cells were seen lying in the subepithelial connective tissue (Fig. 5 ). In all tissues, control sections (PBS or normal mouse serum substituted for the monoclonal antibody step), showed no positive staining.

Quantitation of immunoperoxidase staining Scoring o f samples is shown in Table 1 and the results o f statistical analysis are shown in Table 2. There was a significant effect o f cow ( P < 0.001 ), area o f gland ( P < 0.001 ), t r e a t m e n t ( P < 0.001 ), and t r e a t m e n t - c o w interaction ( P < 0.01 ). The effect o f tissue was not significant ( P > 0.05 ). Further investigation into the effect o f treatment suggested that compared

20

J.L. FITZPATRICK ET AL.

Fig. 4. Teat sinus, immunoperoxidase staining of class II antigens - ovalbumin ( 1 g), infused into this quarter 1 h before slaughter. This section shows dense class II staining (arrows), of the bilayered epithelium (E) of the teat sinus. Cells within the connective tissue (CT) are also densely stained (arrows). X 20.

Fig. 5. Teat duct, immunoperoxidase staining of class II antigens - S . u b e r i s infused into this quarter l h before slaughter. The stratified squamous epithelium (E) and keratin layer (K) of the teat duct show no staining. Class II-positive cells (arrows), are seen accumulated in areas immediately below the epithelium in the subepithelial connective tissue. X 10.

MHC CLASSII EXPRESSIONIN THE COW

21

TABLE 2 Proportion of 20 fields in the heavily stained group I in the epithelium and connective tissue of the gland cistern-secretory tissue junction area of the mammary gland: analysis of deviance when new terms are fitted into the model 2

Cow Area of gland 4 Tissue 5 Treatment s Treatment-cow interaction

Degrees of freedom

Change in deviance

Significance3

8 1 1 3 18

106.367 19.915 0.130 75.076 176.945

P 0.05 P 4.9, P < 0.001 ). The coefficients for ovalbumin or minor pathogens were not statistically significant ( t < 1.51, P > 0.1 ). The sign of the coefficient was positive on both occasions for the minor pathogens, but for ovalbumin it was positive in one model and negative in the other. The significant effect of treatment is reasonably explained in terms of an increase in staining associated with S. uberis infusion. Similarly, the significant effect of area could be explained by an increased intensity of staining at the gland cistern-secretory tissue junction, when compared with the secretory tissue area higher up in the mammary gland. It is concluded, therefore, that infusion ofS. uberis into the mammary gland of dry cows increases MHC class II expression in the epithelial tissue of the gland cistern-secretory tissue area. DISCUSSION

Local intramammary immunisation of the bovine mammary gland produces both a local antibody response (Newby and Bourne, 1977 ), and a local cellular response (Nonnecke et al., 1986). The induction of an immune response involves the activation of T-helper cells and requires the interaction of antigen with class II glycoproteins on the surface of antigen-presenting cells (Unanue and Allen, 1987 ). In this study we have shown that all quarters, including control quarters

22

J.L. FITZPATRICK ET AL.

which were free of bacteria and did not have antigen introduced, contain class II-positive cells in the connective tissue of the mammary gland. Many of these cells had the morphological appearance of macrophages, but in this study no attempt was made to identify them further. The future use of recently available monoclonal antibodies to macrophages, B-cell, T-cells and their subpopulations, in double-staining immunohistology, will permit their characterisation. In contrast, we have also shown that there is little or no constitutive expression of class II molecules by the epithelial cells of the involuted bovine mammary gland. These molecules could, however, be induced by the introduction of dead S. uberis. Infusion with either 1 g or 50 mg ovalbumin, or infection with minor pathogens did not significantly increase class II expression. The role of class II molecules on epithelial cells is unclear. In a review on MHC class II expression by the gut epithelium, Bland ( 1988 ), suggests that the enterocyte may be involved directly in antigen presentation in vivo or may act as an immunological molecular sieve, binding and permitting access only to those peptide ligands to which the individual is able to respond. Similarly, in the mammary gland it is possible that antigen binds to the class II molecules present on the lumenal epithelial cells: the complex may then be endocytosed, hence crossing the epithelium and entering the subepithelium where lymphocytes and macrophages/dendritic cells are concentrated. The antigen could then be presented to cells in association with class II molecules. The expression of class II molecules in response to antigen was patchy in distribution and this suggests a local rather than systemic response. Increased expression was especially evident at the level of the gland cistern-secretory tissue junction in both epithelium and connective tissue. This supports the findings of Nickerson and Heald ( 1982 ), who introduced Staphylococcus aureus into the mammary gland and reported that the maximum concentration of lymphocytes and neutrophils was in the zone closest to the gland cistern. They suggested that this may indicate preferential infiltration of these cells and an attempt to contain the infection in the lower extremities of the gland. Our finding that infusion of dead S. uberis into the dry bovine mammary gland increased the expression of class II molecules in the same area supports a possible immunological role for MHC class II-positive cells. The considerable variation in class II expression between cows following administration of the antigen S. uberis may reflect individual cows' previous experience of the antigen. Further study is required to investigate this and the possible effects of lactational cycle on class II expression in the mammary gland. ACKNOWLEDGEMENTS The authors would like to thank the Agricultural and Food Research Council for their financial support; J.L.F. is in receipt of an AFRC Veterinary Fel-

MHCCLASSII EXPRESSIONINTHECOW

23

1owship. W e are g r a t e f u l t o I L R A D , N a i r o b i , for s u p p l y i n g t h e a n t i - b o v i n e class I I m o n o c l o n a l a n t i b o d y .

REFERENCES Barclay, A.N. and Mason, D.W., 1982. Induction of Ia antigen in rat epidermal cells and gut epithelium by immunological stimuli. J. Exp. Med., 156:1665-1676. Bland, P., 1988. MHC class II expression by the gut epithelium. Immunol. Today, 9:174-178. Bramley, A.J. and Dodd, F.H., 1984. Review of the progress of dairy science. Mastitis control progress and prospects. J. Dairy Res., 51: 481-512. Daar, A.S., Fuggle, S.V., Fabre, J.W., Ting, A. and Morris, P.J., 1984. The detailed distribution of MHC class II antigens in normal human organs. Transplantation, 38: 293-298. Gendelman, H.E., Moench, T.R., Narayan, O. and Griffin, D., 1983. Selection of a fixative for identifying T cell subsets, B cells and macrophages in paraffin-embedded mouse spleen. J. Immunol. Methods, 65: 137-145. Klareskog, L., Forsum, U. and Peterson, P.A., 1980. Hormonal regulation of the expression of Ia antigens on mammary gland epithelium. Eur. J. Immunol., 10: 958-963. Newby, T.J. and Bourne, F.J., 1977. The nature of the local immune system of the bovine mammary gland. J. Immunol., 118: 461-465. Nickerson, S.C. and Heald, C.W., 1982. Cells in local reactions to experimental Staphylococcus aureus infection in bovine mammary gland. J. Dairy Sci., 65:105-116. Nonnecke, B.J., Elsken, L.A. and Kehrli, Jr., M.E., 1986. Local and systemic immune response in the cow after intramammary vaccination during lactation. Vet. Immunol. Immunopathol., 11: 31-44. Skoskiewicz, M.J., Colvin, R.B., Schneeberger, E.E. and Russell, P.S., 1985. Widespread and selective induction of major histocompatibility complex-determined antigens in vivo by gamma interferon. J. Exp. Med., 162: 1645-1664. Unanue, E.R. and Allen, P.M., 1987. The basis for the immunoregulatory role of macrophages and other accessory cells. Science, 236:551-557. Unanue, E.R., Beller, D.I., Lu, C.Y. and Allen, P.M., 1984. Antigen presentation: comments on its regulation and mechanism. J. Immunol., 132: 1-5. Wilson, M.R., 1972. The influence of preparturient intramammary vaccination on bovine mammary secretions. Immunology, 23: 947-955. Wiman, K., Curman, B., Forsum, U., Klareskog, L., Malmnas-Tjernlund, U., Rask, L., Tragardh, L. and Petersen, P.A., 1978. Occurrence of Ia antigens on tissues of non-lymphoid origin. Nature, 276:711-713.

MHC class II expression in the bovine mammary gland.

The distribution of major histocompatibility complex (MHC) class II positive cells within the connective tissue and the epithelium of the involuted bo...
1MB Sizes 0 Downloads 0 Views