Synergism between antibody and neutrophils in the ruminant mammary gland

Veterinary Immunology and Immunopathology, 17 (1987) 389-400 Elsevier Science Publishers B.V., Amsterdam m Printed in The Netherlands

389

SYNERGISM BETWEEN ANTIBODY AND NEUTROPHILS IN THE RUMINANTMAMMARYGLAND

D.L. WATSON CSIRO Division of Animal Health, Armidale, NSW 2350, Australia

ABSTRACT Watson, D.L., 1987. Synergism between antibody and neutrophils in the ruminant mammary gland. Vet. Immunol. Immunopathol., 17: 389-400. Immunological a c t i v i t i e s of the mammary gland are important both as a means of transferring immunity from mother to young and for defending the mammary gland i t s e l f against infection. The presence of immunoglobulins G1, G2 and A, and of neutrophils, macrophages and complement in the ruminant mammary gland is described, in particular the synergistic role of antibody and neutrophils is discussed and studies of immunization against staphylococcal m a s t i t i s are outlined. INTRODUCTION Immunological a c t i v i t y of the mammary gland are of importance for two main reasons.

F i r s t l y , in many species of mammals, ingestion of col ostrum by the

neonate represents the major means of transferring immunity from mother to young (Brambell, 1970). Even in those mammals in which passive immunization occurs before b i r t h

via the placenta or yolk sac endoderm, there is a

substantial degree of anti-microbial protection provided locally in the gut l~men by immunological factors in ingested milk (Goldman, 1973).

Secondly,

immunological a c t i v i t i e s in the mammary tissue and mammary secretion are crucial for defending the mammary gland i t s e l f from infection.

Mastitis is

economically the most important disease of dairy cows throughout the world. Accordingly, considerable

research effort has been directed to gaining an

improved understanding of immunological mechanisms of protection in the mammary gland in devising means by which these may be enhanced and/or exploited to increase resistance to mastitis. In ruminants during the few weeks immediately prepartum, the mammary gland has the unique a b i l i t y of selectively transporting very large quantities of IgGl, from blood

into colostrum (Richards and Marrack, 1963).

Indeed,

im~unoglobulin concentrations in colostrum of the cow may be in excess of 100 mg/ml, most of which is IgG1 (Brandon et a l . , 1971). However, within 1 to 2 days a f t e r p a r t u r i t i o n the levels of immunoglobulin in secretion f a l l dramatically and i t is pertinent, in relation to immunity to mastitis, that 0165-2427/87/$03.50

© 1987 Elsevier Science Publishers B.V.

390 TABLE 1 Immunoglobulin concentrations (mg/ml) in some body fluids of ruminants.

Fluid

I~G

Ovine milk whey1

IgG1

IgG2

0.72

0.08

Caprine milk wheyI

0.25

IgM

IgA

0.03

0.08

0.03

0.06

Bovine milk wheyI

0.35

0.06

0.04

0.05

Ovine jejunal f l u i d I

2.97

1.48

0.30

4.87

Bovine mixed saliva I

0.03

0.01

0.01

0,56

Bovine preputial f l u i d 2

0.59

1,47

0,17

1.17

Ovine bronchial secretion 3

5.65

1,64

0,95

9.51

Data from

I. 2. 3.

Review by Lascelles and McDowell (1974) Winter et al. (1980) Scicchitano et al. (1986)

immunoglobulin levels in normal milk are quite low, especially in comparison with concentrations in other body fluids which bathe epithelial membranes (except saliva) (Table 1). The cells present in mammary secretions of ruminants may vary considerably in both type and number depending on the stage of lactation.

In colostrum,

neutrophils are the predominant cell type but in normal milk the majority of cells are macrophages; in the early stages of involution the neutrophil

is

again the major cell type present but when the gland has completely involuted macrophages predominate (Lee and Outteridge, 1981; McDonald and Anderson, 1981) (Table 2).

Bacterial infection of the mammary gland at any stage of lactation,

however, results in the appearance in mammary tissue and secretion of very large numbers of neutrophils which migrate from the blood vessels serving the gland (Jain, 1976). IgA AND THE LOCAL IMMUNERESPONSE IN THE MAMMARYGLAND IgA was discovered in 1959 (Heremans et a l . , 1959) and in general i t

is

established as the major immunoglobulin isotype in the mucous secretions and mammary secretions of most species of animals (Tomasi and Bienenstock, 1968). However, i t is important that the limitations of this generalization be recognized. In the mammary secretions of ruminants, IgA is quantitatively a

391

TABLE 2 Differential counts (%) of leucocytes in bovine mammary secretions.

Leucocyte

Colostrum

Neutrophil

Milk

Dry gland secretion

62

3

3

25 10

15 65

21 68

Lymphocytes

4

16

7

Epithelial cells

0

2

1

Macrophages - vacuolated non-vacuolated -

Data are mean values for 4 cows derived from results of Lee et al. (1980). r e l a t i v e l y minor isotype (Table 1) (Lascelles and McDowell, 1974). Local antigenic stimulation of the preparturient mammarygland, however, i n i t i a t e s a local

immune response and as a result a considerable

proportion

of the

immunoglobulin in milk from locally immunized glands is IgA (Lascelles and McDowell, 1970; Watson and Lascelles, 1973).

The crucial question is how, i f

at a l l , does this IgA mediate immune protection? ruminant species (but l i t t l e

There is evidence for non-

for ruminant) that milk IgA with anti-bacterial

(Wilson and Svendsen, 1971) or a n t i - v i r a l a c t i v i t y (Sail and Bohl, 1981) may confer to the suckling neonate protection from enteric diseases.

I t is thought

that milk IgA lying in the mucous blanket of the intestine binds to the virus or bacteria thereby 'blocking'

adherence of the pathogen to intestinal

epithelial cells (Rutter et a l . , 1976).

I t is possible that IgA antibody in

milk could similarly prevent adherence of bacteria to the epithelium of the mammary gland although i t

should be pointed out that

the involvement of

bacterial adherence in the pathogenesis of mastitis is an issue about which there remains considerable conjecture

(Frost,

1975; Anderson,

1978a).

A l t e r n a t i v e l y , or additionally, milk IgA may neutralize toxins produced by microbial pathogens. That IgA mediates protection by acting synergistically with cells in milk is a possibility worthy of investigation.

I t has been shown recently that IgA is

a potent mediator of antibody-dependent cellular cytotoxicity in a murine system in which ShiBella organisms were used as the targets and lymphocytes from gut-associated lymphoid tissue (GALT) were the effector cells (Tagliabue et a l . , 1983).

Or particular importance was the finding that IgA had no anti-

bacterial effect when lymphocytes derived from popliteal lymph nodes or thymus were used in the assay.

The evidence presently a v a i l a b l e for

ruminants

392

suggests that

IgA is u n l i k e l y to exert any immune protection in the mammary

gland through an association with neutrophils. have membrane receptors for IgA (Fanger et a l . ,

Although human neutrophils may 1980) neutrophils from sheep,

goats and c a t t l e almost c e r t a i n l y do not (Watson, 1975; Micusan and Borduas, 1977; McGuire et a l . ,

1979).

Furthermore IgA antibody has been implicated in

the impairment of neutrophil-mediated phagocytosis of microorganisms (Wilton, 1978; Magnusson et a l . ,

1979).

Nevertheless i t

may play a role in protection

is possible that IgA antibody

of the mammary gland

in

association

with

macrophages, lymphocytes or even secretory e p i t h e l i a l c e l l s , which in mice at least appear to be able to phagocytose bacteria (Chandler et a l . , 1980). Finally it

should be mentioned t h a t , in addition to stimulating production

of secretory IgA, local immunization of the mammary gland "primes" t h i s organ for an enhanced inflammatory response upon subsequent challenge (Targowski and Berman, 1975; Colditz and Watson, 1982). characterized

by the

earlier

arrival

Such an inflammatory respones is

of larger numbers of neutrophils

in

mammary tissue and secretion than is the case for non-immunized mammary glands. This is p a r t i c u l a r l y relevant as i t is known that an important factor in determining the s e v e r i t y of m a s t i t i s is the speed with which an animal can mobilize neutrophils to the infected tissue ( H i l l , 1981). THE ROLE OF NEUTROPHILS IN MAMMARYGLAND DEFENCE There is compelling evidence in favour of the p r o p o s i t i o n that the neutrophil is the key host factor in c o n t r o l l i n g bacterial infection of the mammary gland (reviewed by Jain, 1976; Colditz and Watson, 1985). subclinical

staphylococcal

mastitits,

e x p e r i m e n t a l l y induced

In cows with neutropenia

resulted in conversion of the disease to the acute-gangrenous form (Schalm et al.,

1976).

In c o n t r a s t ,

pre-existing

neutrophilic

leucocytosis

in milk

impeded the otherwise successful establishment of Streptococcus a~alactiae in the l a c t a t i n g mammary gland (Schalm et a l . , 1966). The numbers of neutrophils in normal milk are quite low (around 3000/ml) compared with blood (2 x 106/mi), however there is rapid migration of these c e i l s from blood through the parenchyma of the gland and into milk when an infection occurs.

Neutrophil numbers in milk begin increasing within 4 hours

) f infusion of Staphylococcus aureus into the l a c t i f e r o u s sinus and by 24 hours post-infection t h e i r concentration may be >107/ml (Watson, 1984). The primary function of neutrophils is phagocytosis of "foreign" material. Neutrophils which have migrated into milk appear to engage aggressively in phagocytosis of bacteria. Indeed there is evidence that neutrophils which have migrated in response to chemotactic stimuli have enhanced phagocytic c a p a b i l i t y compared with neutrophils in blood (Van Epps and Garcia, 1980). On the other hand neutrophils a r r i v i n g in milk may also phagocytose casein (Russell and

393

Reiter, 1975) and milk fat globules (Paape et a l . , 1975), an activity which lowers the overall antimicrobial effectiveness of these cells in the gland. Neutrophils may phagocytose bacteria in the absence of opsonins.

This

process occurs through interaction between l e c t i n - l i k e receptors on the neutrophil membrane and carobhydrate ligands on the cell wall of bacteria (Glass et a l . , 1981). Howeverneutrophils bear membrane receptors for opsonins (C3 and immunoglobulins) and the efficiency of phagocytosis in the presence of these molecules is greatly increased. C3b may act as an opsonin when antibody binds to microbial antigen and initiates either the classical or alternative pathways of complement fixation (Klebanoff and Clark, 1978). Additionally cell wall constituents of many microorganisms can activate complement via the alternative pathway (Williams and Quie, 1971).

In ruminants haemolytic

complement is present in colostrum and involution secretion but is not detectable or present only at extremely low concentrations in normal milk (Reiter and Oram, 1967; Eckblad et a l . , 1981; Mueller et a l . , 1983). These findings suggest that complement is not an important opsonin in the lactating gland until inflammation occurs and leakage of intravascular protein into the secretion becomes pronounced.

However, complement may play an important

opsonic role during colostrogenesis and early involution when both C3 and neutrophils are present in secretions at significant levels. Neutrophils have membrane receptors for the Fc region of IgG molecules and in sheep, goats and cattle the neutrophil receptor is specific for the IgG2 subclass (Watson, 1975; Micuson and Borduas, 1977; McGuire et a l . , 1979). Mammary neutrophils carry cytophilic IgG2 on the cell membrane and under in vivo conditions there appears to be f a i r l y low a f f i n i t y between molecule and receptor

(Watson, 1975).

The presence of cytophilic IgG2 of appropriate

antibody specificities presumably enhances the immunological surveillance capabilities of the neutrophil.

When cytophilic IgG2 binds to microbial

antigen, or when the receptor of the neutrophil interacts with the Fc region of IgG2 complexed with microbial antigen, the association between immunoglobulin and cell membrane becomes one of strong a f f i n i t y and appears to signal the initiation of active ingestion by the cell (Leslie and Alexander, 1979).

I t is

worth noting that although opsonization with C3 mediates effective attachment between neutrophil and microbe i t stimulates ingestion at only a "basal" rate. Opsonization with IgG, however, initiates an "active" ingestion process by the cell, resulting in more rapid and efficient phagocytosis of microbial prey (Hed and Stendahl, 1982). In addition to direct opsonization by IgG2 antibody, complement fixation may be mediated by IgG1, IgG2 and IgM antibody each of which f i x homologous complement (Feinstein and Hobart, 1969; Hobart, 1976). Thus antibody of isotypes for which neutrophils do not have receptors may nevertheless promote

394

phagocytosis via C3b opsonization. D a t a From Williams and Hill (1982) indicated that bovine IgM may play a role in opsonizing E. coli and S. aureus in the absence of complement, but as these authors failed to demonstrate IgM receptors on bovine neutrophils the precise mechanism by which this might occur is unknown. for

both

I t should be added that bovine macrophages bear membrane receptors

IgG1 and IgG2 (Rossi

and Kiesel, 1977) and these cells have a

prominent phagocytic role in the mammary gland (Desiderio and Campbell, 1980). Various studies have been concerned with the effectiveness of milk as a source of opsonins for phagocytosis of mastitis pathogens (Paape et a l . , 1975; Hill et a l . , 1978) and several workers have drawn the conclusion that milk is an adequate source of opsonins for neutrophil-mediated phagocytosis (Brock et a l . , 1975; Williams and Bunch, 1981; Anderson and Williams, 1985). However, in vitro phagocytosis experiments can produce deceptive results which may lead to erroneous conclusions about in vivo neutrophil-mediated protection of the mammary gland.

In particular many of these experiments ignore the virulence

properties and adaptive capabilities of the mastitis pathogen. Staphylococcus aureus, for example, when growing in the mammary gland expresses a capsule (Norcross and Opdebeeck, 1983) or pseudocapsule (Watson, unpublished results) around the cell wall.

The pseudocapsule is a powerful virulence determinant

which confers on the bacteria considerable resistance to phagocytosis (Karakawa et a l . , 1978; Watson, 1982) (Table 3), explain t h i s

There are at least two mechanisms to

antiphagocytic phenomenon.

F i r s t l y , "surface phagocytosis",

mediated through the l e c t i n - l i k e receptors on the neutrophil membrane, is

TABLE 3 Results of phagocytosis assays employing ovine mammary neutrophils as effector c e l l s , serum from sheep immunized with Staphylococcus aureus vaccines as opsonin and S. aureus grown under in v i t r o or in vivo c o n ~ n s as target cells. Data are mean numbers of surviving staphylococci colony forming units (from Watson, 1982).

Opsonin Serum from sheep immunized with:

S. aureus cultural conditions In v i t r o

In vivo

Killed vaccine A + FIA*

2,986

20,235

Killed vaccine B + FIA

3,049

11,780

Live vaccine

3,964

4,114

*Freund's Incomplete Adjuvant

395

prevented because complementary ligands on the bacterial cell wall are shrouded by pseudocapsule. Secondly, activation of the alternative complement pathway by peptidoglycan in the cell wall results in binding of C3b beneath the capsule/pseudo-capsule, a location which makes i t

inaccessible to the C3b

receptor on the neutrophil membrane (Verbrugh et a l . , 1980, 1982).

However

specific IgG anti-capsular antibody can neutralize the antiphagocytic effect of the capsule/pseudocapsule. In assessing neutrophil-mediated defence of the udder i t is important not to become unduly pre-occupied with opsonins in milk from 'normal', non-immunized animals since i t

is known that such animals usually develop mastitis i f

v i r u l e n t pathogens gain access to the gland.

An important

practical

requirement is to devise means of increasing the levels of effective opsonins in milk before, or soon after, infection occurs.

In this context "effective

opsonins" are antibodies with specific activity directed against epitopes on the bacterial pseudocapsule and belonging to an immunoglobulin isotype for which phagocytes in the gland have Fc receptors. The presence in the gland of specific antibody with potent opsonizing activity is not only important for enhancing phagocytosis by neutrophils but also for promoting more efficient intracellular k i l l i n g of engulfed bacteria. In contrast to the relatively benign ingestion mediated by occupation of the C3b receptor alone, ingestion initiated through the Fc receptor triggers the "metabolic burst" by the neutrophil (Christie et a l . , 1976; Hed and Stendahl, 1982). VACCINATION AGAINST STAPHYLOCOCCAL MASTITIS IN RUMINANTS There have been a great number of attempts over the past 100 years to immunize ruminants against staphylococcal mastitis.

Most of these were

unsuccessful or met with only limited success (Singleton et a l . , 1967; Brock et a l . , 1975). However, almost 80 years ago i t was shown that the subcutaneous injection of live S. aureus organisms into ewes resulted in a substantial level of resistance from experimental staphylococcal mastitis (Bridre, 1907). More recently i t

was confirmed that live S. aureus vaccines confer significant

protection from experimental mastitis in ewes whereas conventional, k i l l e d vaccines do not (Watson and Lee, 1978). Studies in our laboratory have provided some understanding of the mechanisms which appear to be responsible for this observed protection. Injection of live staphylococcal

vaccines results in the production of considerably greater

levels of IgG2 antibody than does vaccination with killed staphylococci. IgG2 is cytophilic for neutrophils and is a potent opsonin in phagocytosis assays using S. aureus targets (Watson, 1976). The particular opsonic effectiveness of this IgG2 results from the fact that much of the antibody activity is

396

directed

against

"in

vivo antigens" embedded in the pseudocapsule of the

bacteria; these IgG2 opsonins counteract the otherwise antiphagocytic effect of the pseudocapsule (Watson and Prideaux,

1979; Watson, unpublished results).

Furthermore, l i v e S. aureus vaccines predicate a high ratio of IgG2:IgG1, for both s p e c i f i c

antibody and total

IgG, during the humoral immune response

whereas for k i l l e d staphylococcal vaccines the reverse is the case (Kennedy and Watson, 1982; Kerlin and Watson, 1986).

Finally, in ewes at least, l i v e S.

aureus vaccines "prime" the animal for a more rapid mobilization of neutrophils into mammary secretion i f the gland is subsequently infected with staphylococci (Colditz and Watson, 1982, 1984). In the USA considerable

attention has been given to the p o s s i b i l i t y of

potentiating udder deFence by introducing a s t e r i l e

polyethylene loop or

intramammary device (IMD) through the teat duct into the gland cistern.

The

effect of the IMD is to provoke a chronic neutrophilic leucocytosis in the gland with concomitant increases in levels of serum proteins in milk (Paape et a l . , 1981). Cows f i t t e d with the IMD have increased resistance to infection with S. aureus (Paape et a l . ,

1981) however i t

seems l i k e l y that protection

afforded by the device could be improved even further i f

i t s use were to be

combined with syste~nic administration of an effective staphpylococcal vaccine, with a resultant increase in levels of potent antibody opsonins in milk. Although other deFence mechanisms may be invoked in future strategies for immunization against mastitis there is l i t t l e doubt that successful vaccination (at least against gram-positive bacteria) cannot be achieved without u t i l i z i n g the resources of both neutrophils and opsonins.

Such a strategy has provoked

some debate (semantic and philosophical) in which i t

is argued that " . . . i t

becomes evident that the natural defence mechanism of the mammary gland cannot be used, whether or not enhanced by immunization, to prevent mastitis since i t is the defence system that is defined as the disease" (Anderson, 1978b). Based on our current knowledge of pathogenesis of staphylococcal mastitis and immune mechanisms in the mammary gland, i t

is inevitable that vaccinated ("immune")

animal suffer a substantial, though transient, neutrophilic leucocytosis in milk i f infected with S. aureus. result

is

immeasureably

However, they recover from the disease.

This

preferable to the consequences of infection in

unvaccinated animals. CONCLUSIONS There has been a tendency over the past 10 years to include the mammary gland in "the common mucosal immune systen" (Bienenstock and Befus, 1980). This has resulted from the finding, in some species, that immunocytes committed to IgA synthesis may leave GALT and relocate in mammary tissue (Goldblum et al.,

1975).

However, the epithelium of the mammary gland is not a mucous

397

membrane nor does there appear to be any collateral relationship between GALT and the immune system of the ruminant mammary gland (Sheldrake et a l . , 1986), Even in those species in which there is a gut-mammary gland axis the receptiveness of mammary tissue for IgA-producing cell precursors is under hormonal regulation (Weisz-Carrington et a l . , 1978). In ruminants IgG is the i mmunoglobulin isotype of major importance in mammary secretions.

IgG1 is selectively transported from blood into colostrum

and is largely responsible for immune protection of the suckling neonate. IgG2 occurs in milk in very low concentrations but may enter the section as cytophilic immunoglobulin on the cell membrane of neutrophils and by passive transudation from blood when inflammation of the gland occurs. I t is established that IgG2 is a potent opsonin for neutrophil-mediated phagocytosis of staphylococci in sheep and cattle and the synergistic a c t i v i t y of IgG2 antibody and neutrophils is important in protecting the mammary gland from experimental staphylococcal mastitis. REFERENCES Anderson, J.C., 1978a. Absence of bacterial adherence in the establishment of experimental mastitis in mice. Vet. Pathol., 15: 770-775. Anderson, J.C., 1978b. The problem of immunization against staphylococcal mastitis. Br. vet. J., 134: 412-420. Anderson, J.C. and Williams, M.R., 1985. The contribution of a capsule to survival of staphylococci within bovine neutrophils. J. med. Microbiol., 20: 317-323. Bienenstock, J. and Befus, A.D., 1980. Mucosal immunology. Immunology, 41: 249-270. Brambell, F.W.R., 1970. The transmission of passive immunity from mother to young. In: A. Neuberger and E.L. Tatum (Editors), Frontiers of Biology, Vol. 18. North-Holland, Amsterdam. Brandon, M.R., watson, D.L. and Lascelles, A.K., 1971. The mechanism of transfer of immunoglobulin into mammary secretion of cows. Aust. J. exp. Biol. med. Sci., 49: 613-623. Bridre, J., 1907. La mammite gangreneuse des brebis l a i t i e r e s : pathogenie et vaccination. Bull. Soc. Cent. Med. Vet., 61: 500-506. Brock, J.H., Steel, E.D. and Reiter, B., 1975. The effect of intramuscular and intramammary vaccination of cows on antibody l e v e l s and resistance to intramammary infection by Staphylococcus aureus. Res. vet. Sci., 19: 152158. Chandler, R.L., Smith, K. and Turfrey, B.A., 1980. Studies on the phagocytic potential of secretory epithelial cells in experimental mastitis. J. Comp. Path., 90: 385"394. Christie, K.E., Solberg, C.O., Larsen, B.O., Grov, A. and Tender. 0., 1976. Influence of IgG, F(ab)2 and IgM on the phagocytic and bactericidal a c t i v i t i e s of human neutrophil granulocytes. Acta Path. Microbiol. Scand. C., 84: 119-123. Colditz, I.G. and Watson, D.L., 1982. Effect of immunization on the early influx of neutrophils during staphylococcal mastitis in ewes. Res. vet. Sci., 33: 146-151. C o l d i t z , I.G. and Watson, D,L., 1984. The role of humoral and cellular mediators in enhanced mammary inflammatory reactions to staphylococcal infection in systemically immunized ewes. Microbiol. Immunol., 26: 11711180.

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