Journal of Mammary Gland Biology and Neoplasia, Vol. 7, No. 2, April 2002 ( C 2002) Mammary Gland Immunity and Mastitis Susceptibility Lorraine M. Sordillo1,2 and Katie L. Streicher1 Lactation is considered the ﬁnal phase of the mammalian reproductive cycle, and the mammary gland provides milk for nourishment and disease resistance to the newborn. However, the cellular and soluble immune components associated with mammary tissues and secretion also can play an important role in protecting the gland from infectious diseases, such as mastitis. Mastitis can affect essentially all lactating mammals, but is especially problematic for dairy cattle. The most recent estimates from the National Mastitis Council suggest that mastitis affects one third of all dairy cows and will cost the dairy industry over 2 billion dollars annually in the United States in lost proﬁts (National Mastitis Council (1996) Current Concepts in Bovine Mastitis, National Mastitis Council, Madison, WI). The overall impact of mastitis on the quality and quantity of milk produced for human consumption has provided the impetus to better understand the pathophysiology of the mammary gland and develop ways to enhance disease resistance through immunoregulation. As such, the bovine species has played a critical and prominent role in our current understanding of mammary gland immunobiology. This paper provides a comprehensive overview of mammary gland immunity and how the stage of lactation can impact important host defenses. While this review emphasizes the bovine system, comparisons to humans and other domestic mammals will be addressed as well. KEY WORDS: mammary immunology; mastitis; neutrophils; lymphocytes; cytokine. INTRODUCTION factors that attempt to eliminate the foreign organism, and subsequent exposure to the same pathogen in- Through genetic selection and technological ad- duces a more pronounced response. In contrast, non- vances in milk removal, the bovine mammary gland immune components of mammary gland immunity yields far more milk than is needed to nourish the consist of a variety of disease-resistance mechanisms newborn calf. Factors associated with the intense man- that are not speciﬁc to a particular pathogen and are agement of dairy cattle can profoundly affect mam- not augmented by repeated exposure to the same for- mary gland defense mechanisms and the ability of the eign molecule. While both immune and nonimmune host to resist mastitis. Mammary gland immunity, de- factors interact extensively to provide adequate pro- ﬁned as the protection and resistance to infectious dis- tection against mastitis, the magnitude, duration, and ease, is facilitated through a variety of immune and effectiveness of mammary gland immunity are inﬂu- nonimmunological factors. The immune response is enced greatly by particular etiological agents. characterized by the ability to recognize and discrim- Technological advances in immunology have inate between foreign substances and the body’s own provided new research tools that have facilitated molecules. Upon recognition of invading pathogens, the study of mammary gland immunity and disease the immune system enlists both cellular and soluble pathogenesis. As a consequence, the last two decades 1 Department of Veterinary Science, The Pennsylvania State Uni- Abbreviations used: CSF, colony stimulating factor; GH, growth versity, University Park, Pennsylvania. hormone; IGF, insulin-like growth factor; IFN, interferon; IL, in- 2 To whom correspondence should be addressed at 115 Henning terleukin; Ig, immunoglobulin; MHC, major histocompatibility Building, The Pennsylvania State University, University Park, complex; NK, natural killer; TNF, tumor necrosis factor; SCC, Pennsylvania 16870. somatic cell count; IMI, intramammary infection. 135 1083-3021/02/0400-0135/0 C 2002 Plenum Publishing Corporation 136 Sordillo and Streicher have seen major progress in understanding the bovine diminished mammary gland defense mechanisms (8). mammary gland defense system and its function in It is interesting to note that other mammals, includ- preventing disease. In the 1980s, considerable atten- ing women, also suffer from increased susceptibility to tion was focused on the identiﬁcation and functional mastitis during the immediate postpartum period (9). characterization of bovine mammary gland leuko- However, changes in mammary gland defense mecha- cyte populations. Different leukocyte subpopulations nisms during the periparturient period and the subse- were conveniently identiﬁed by ﬂow cytometry us- quent impact on susceptibility to new intramammary ing monoclonal antibodies developed to recognize infections have been characterized more extensively unique surface molecules. Since 1990, this research for the bovine than any other mammal. As such, the has extended to the study of bovine cytokines and bovine species has played a crucial role in illuminat- their potential roles in the pathophysiology and con- ing our current understanding of mammary gland im- trol of mastitis. The availability of gene and pro- munobiology as it relates to bacterial infections. The tein sequences led to the development of several challenge that confronts researchers now is to gain a strategies to identify these cytokines, including mono- better understanding of the complex interactions be- clonal antibodies, primers for polymerase chain reac- tween the pathogenesis of particular bacteria, host tions, and quantiﬁcation with bioassays and ELISA. responses needed to eliminate the pathogens from Moreover, recombinant cytokines were produced to the mammary gland, and ways to enhance mammary explore their immunomodulatory and therapeutic gland immunity before disease is established. uses for disease control. Mammary gland immunity is a rapidly evolving ﬁeld of research in veterinary medicine. Therefore, it is most opportune to review DEFENSE MECHANISMS the current knowledge of various immune and nonim- mune components of host defense and the underlying The mammary gland is protected by a variety of mechanisms of disease resistance. defense mechanisms, which can be separated into two distinct categories: innate immunity and speciﬁc im- munity. Innate immunity, also known as nonspeciﬁc SUSCEPTIBILITY TO MASTITIS or nonimmune responsiveness, is the predominant de- fense during the early stages of infection. Nonspeciﬁc The incidence of mastitis increases when defense responses are present or are activated quickly at the mechanisms of the mammary gland are impaired. site of infection by numerous stimuli, but are not aug- Dairy cattle are exposed to numerous genetic, physi- mented by repeated exposure to the same insult. If ological, and environmental factors that can compro- nonspeciﬁc defense mechanisms function adequately, mise host immunity and increase the incidence of mas- most pathogens are readily eliminated within a short titis (1,2). Emphasis on genetic selection to maximize period of time and before the speciﬁc immune system milk production has increased metabolic stresses as- is activated. Rapid elimination of bacteria often will sociated with milk synthesis and secretion, and a nega- not result in noticeable changes in milk quality or pro- tive correlation exists between milk production capac- duction. Nonspeciﬁc or innate defenses of the mam- ity and resistance to mastitis (3). Therefore, it is likely mary gland are mediated by the physical barrier of that resistance to mastitis will deteriorate in dairy cat- the teat end, macrophages, neutrophils, natural killer tle populations that are under selection for improved (NK) cells, and certain soluble factors. milk production. Removal of milk by milking ma- If a pathogen is able to evade or is not completely chines can cause trauma of teat end tissues which facil- eliminated by the innate defense system, the speciﬁc itates colonization by mastitis-causing organisms. To- or acquired immune system is triggered. Acquired tal conﬁnement housing, increased cow densities per immune responses recognize speciﬁc antigenic deter- unit area, and use of bedding materials that support minants of a pathogen. If a host should encounter bacterial growth also can have a marked impact on the same antigen more than once, a heightened state the susceptibility of dairy cattle to mastitis by over- of immune reactivity would occur as a consequence whelming important local defense mechanisms (4). of immunological memory. In comparison with the However, one of the more signiﬁcant factors known to ﬁrst exposure to a bacterial antigen, a memory re- inﬂuence mammary gland defense capabilities is lac- sponse would be much faster, considerably stronger, tation stage (5–7). Dairy cattle are especially suscepti- longer lasting, and often more effective in clearing the ble to mastitis during the periparturient period due to pathogen. Vaccination protocols are based on these Immunobiology of the Mammary Gland 137 unique features of the speciﬁc immune response. The increased intramammary pressure (8,13). The mam- immune system is able to distinguish self from nonself mary gland is especially susceptible to mastitis during and selectively react only to foreign antigens through this time, partially due to the dilation of the teat canal genetically diverse, membrane-bound proteins called and leakage of mammary secretions. major histocompatibility complex (MHC) molecules. A speciﬁc immune response only will occur if anti- gens are combined with an MHC molecule on the Cellular Defenses surface of certain cells, a process referred to as anti- gen presentation. Recognition of pathogenic factors If bacteria are able to overcome the anatomical for elimination is mediated by macrophages, several defense provided by the teat end, they still must evade lymphoid populations, and immunoglobulins (Ig) or the antibacterial activities of the mammary gland in antibodies. order to establish disease. The total number and ac- In the mammary gland, it is necessary for both in- tivity of mammary gland leukocyte populations play nate and acquired immunity to be highly interactive early and vital roles in determining the severity and and coordinated in order to provide optimal protec- duration of intramammary infections. Resident and tion from mastitis. Because of the highly integrated newly recruited mammary leukocytes consist of sev- nature of mammary defense mechanisms, this review eral types, including neutrophils, macrophages, and will further characterize mammary immune factors lymphocytes that are capable of mediating either in- into anatomical, cellular, and soluble components and nate or acquired immune responses (see Table I). discuss important changes that occur in host defense Neutrophils are nonspeciﬁc leukocytes that are during the periparturient period. actively recruited to the site of infection and are the principal cell type found in mammary tissues and secretions during the early stage of the inﬂammatory Anatomical Defenses Table I. Summary of Mammary Gland Cellular Defenses The teat end is considered the ﬁrst line of de- Factor Biological function fense against mastitis since this is the route by which invading pathogens can gain entrance to the mam- Neutrophils Phagocytosis and intracellular killing of mary gland. The teat contains sphincter muscles that bacteria; secretion of antibacterial maintain tight closure between milkings and hin- factors Macrophages Phagocytosis and intracellular killing of der bacterial penetration. Increased patency of the bacteria; antigen presentation in sphincter is directly related to increased incidence of conjunction with MHC mastitis. The teat canal also is lined with keratin, which Natural killer cells Nonimmune lymphocytes that secrete is crucial to the maintenance of the barrier function antibacterial proteins upon activation of the teat end; removal of the keratin has been cor- T lymphocytes CD4+ (T helper) Production of immunoregulatory related to increased susceptibility to bacterial inva- cytokines following antigen sion and colonization. Teat keratin is a waxy material recognition with MHC class II that is derived from stratiﬁed squamous epithelium. molecules; memory cells following Accumulation of keratin can provide a physical ob- antigen recognition struction to bacteria, hindering their migration into CD8+ (T cytotoxic) Lysis of altered or damaged host cells when complexed with MHC class I the gland cistern. In fact, the keratin structure can molecules; production of cytokines completely occlude the duct during the nonlactating that can down-regulate certain period (10). Within the keratin lining, antimicrobial leukocyte functions agents have been identiﬁed (11). The esteriﬁed and γ δ T lymphocytes Biological role in the mammary gland nonesteriﬁed fatty acids present in teat keratin are is speculative B lymphocytes bacteriostatic and include myristic acid, palmitoleic Mature B cells Display membrane-bound antibody acid, and linoleic acid. Cationic proteins associated molecules to facilitate antigen with the keratin lining can bind electrostatically to presentation; memory cells following mastitis pathogens, alter their cell walls, and render antigen interactions them more susceptible to osmotic pressure (11,12). As Plasma cell Terminally differentiated B lymphocytes that synthesize and secrete antibody parturition approaches, considerable ﬂuid accumula- against a speciﬁc antigen tion occurs within the bovine mammary gland, causing 138 Sordillo and Streicher process associated with bacterial infection (14). While development of speciﬁc immune responses through neutrophil numbers are relatively low in the healthy antigen processing and presentation in association mammary gland (<105 cells/mL), their numbers can with MHC class II antigens (26,27). constitute greater than 90% of the total mammary Dramatic alterations in the functional capabili- leukocyte population during mastitis (>106 cells/mL). ties of mammary gland macrophages occur during the These nonspeciﬁc cells travel from the blood to the periparturient period and have been directly linked mammary gland in response to a variety of inﬂamma- with disease incidence. Although bovine macrophage tory mediators in order to phagocytose and kill bac- numbers are highest in the last week of gestation, terial pathogens (15). Neutrophils have bactericidal the phagocytic capacity of these cells is decreased, effects that are mediated through a respiratory burst possibly due to the lower opsonic activity in mam- that produces hydroxyl and oxygen radicals (16). In mary secretions. This decrease could be mediated by addition, neutrophils are a source of small antibac- a decrease in IgM, which facilitates phagocytosis by terial peptides, defensins, which can kill a variety of both macrophages and neutrophils (2). Similar to the mastitis-causing pathogens (17). During the peripar- bovine system, the opsonic activity of components turient period, a number of neutrophil functions are within porcine mammary secretions was signiﬁcantly altered or impaired (5,16,18,19). At this time the num- lower during the periparturient period, which could ber of immature neutrophils in the bovine blood in- lead to less efﬁcient phagocytosis (28). Additionally, creases, while the number of mature neutrophils in MHC II expression by bovine macrophages during the blood and mammary secretions is lowest. Neu- the peripartum period is decreased, which could con- trophil populations also exhibit impaired function of tribute to poor antigen presentation and result in major defense-related activities, such as phagocyto- a weaker speciﬁc immune response from mammary sis, respiratory burst activity, superoxide anion pro- gland lymphocytes (6,26). duction, random cellular migration, and chemotaxis, Lymphocytes are able to recognize antigens around parturition (5). Chemotactic neutrophil mi- through speciﬁc membrane receptors which deﬁne gration from blood could be impaired further due to the immunological characteristics of speciﬁcity, diver- the decreased proportion of cells expressing the ad- sity, memory, and self/nonself recognition. Subsets of hesion receptor CD62L (L-selectin) that is necessary lymphocytes are divided into two main groups: T and for penetration through the endothelium to sites of B lymphocytes. The T lymphocytes can be classiﬁed infection (20). further into αβ T lymphocytes, which include CD4+ Macrophages represent the dominant cell type (T helper) and CD8+ (T cytotoxic or T suppres- seen in milk and tissues of healthy, lactating mammary sor) lymphocytes, and γ δ T cells. In healthy human, glands. During bacterial pathogenesis, macrophages porcine, and bovine mammary glands, αβ T lympho- may serve to facilitate either innate or acquired im- cytes prevail and predominantly express the CD8+ mune responses. Similar to neutrophils, the nonspe- phenotype, in contrast to peripheral blood, which has ciﬁc functions of macrophages are to phagocytize primarily the CD4+ phenotype (7,29,30). Lymphoid bacteria and destroy them with proteases and re- cells in both human and bovine milk also display active oxygen species. The phagocytic rate of these a memory cell phenotype (31,32). However, CD4+ cells can be increased dramatically in the presence lymphocytes are the most prominent cell type at all of opsonic antibodies against particular pathogens lactation stages in caprine mammary glands (33). De- (21,22). However, mammary macrophage numbers pending on the stage of lactation and tissue location, tend to be lower during inﬂammation and they pos- the percentages of lymphocyte subsets can vary sig- sess fewer Fc receptors, possibly decreasing their rate niﬁcantly and major shifts in trafﬁcking patterns are of phagocytosis when compared to neutrophils (23). correlated with susceptibility to disease (7,25,34). Therefore, the ability of macrophages to secrete sub- During mastitis, CD4+ T lymphocytes prevail stances that facilitate the migration and bacterici- and are activated in response to recognition of dal activities of neutrophils is believed to be of antigen-MHC class II complexes on antigen- greater importance to the nonspeciﬁc defense of the presenting cells, such as B cells or macrophages. mammary gland than acting as professional phago- These cells function to activate lymphocytes and cytes. Indeed, activated macrophages are triggered macrophages by their ability to secrete certain cy- to release prostaglandins, leukotrienes, and cytokines tokines. Depending on the repertoire of cytokines that can greatly augment local inﬂammatory pro- produced, the T helper cell response can facili- cesses (15,24,25). Macrophages also play a role in the tate either a cell-mediated (Th1 type) or a humoral Immunobiology of the Mammary Gland 139 (Th2 type) immune response (35). While the initial in a non-MHC-restricted way (41). The cytotoxic abil- characterization of the Th1 vs. Th2 paradigm in mice ity of γ δ T lymphocytes suggests that they may be able is now considered somewhat oversimpliﬁed, inter- to destroy altered epithelial cells, including malignant, leukin (IL)-2 and interferon (IFN)-γ were character- breast carcinoma cell lines (42). These cells also may ized as the major cytokines produced during the Th1 play a role in infectious diseases and therefore provide response and IL-4, IL-5, and IL-10 dominate during an important line of defense against bacterial infec- the Th2 response. However, it is known that IL-10 tions. Relative to the blood, both humans and rumi- can be produced by and regulate all subtypes of Th nants express greater levels of γ δ T lymphocytes in cells (36). During the periparturient period, CD4+ mammary secretions and parenchyma (43). The con- cells produce less IL-2 and IFN-γ , but more IL-4 and tributions of γ δ T cell variations to overall mammary IL-10, compared to CD4+ cells obtained during later immunity are not as clear because the biological func- stages of lactation (37). The signiﬁcance of the shift tions of γ δ T cells are still speculative. to a Th2-type response on disease susceptibility in the The primary role of B lymphocytes is to pro- mammary gland has not been deﬁned. duce antibodies against invading pathogens. Unlike The CD8+ T cells have either a cytotoxic or macrophages and neutrophils, B lymphocytes uti- suppressor function, such that they either eliminate lize their cell surface receptors to recognize speciﬁc host cells expressing foreign antigen (in association pathogens. Similar to macrophages and dendritic cells, with MHC class I) or they control the immune re- B lymphocytes can function as antigen-presenting sponse by suppressing the activation of these cells cells as they internalize, process, and present anti- during bacterial infections. It has been suggested that gen in the context of MHC class II molecules to T- CD8+ cytotoxic cells may act as scavengers, remov- helper lymphocytes. Upon presentation of the pro- ing old or damaged secretory cells, the presence of cessed antigen, IL-2 is secreted by the T lymphocytes, which could increase the susceptibility of the mam- which in turn induces proliferation and differentia- mary gland to infections (32). Suppressor T lympho- tion of the B lymphocyte into either plasma cells that cytes are thought to control or modulate the im- produce antibody or memory cells. Unlike T lympho- mune response to bacterial infections. Researchers cytes, the percentages of B lymphocytes remain fairly have demonstrated that CD8+ lymphocytes activated constant between stages of lactation (7). during bacterial infections can suppress important Natural killer (NK) cells are large, granular lym- host immune responses. Evaluation of lacteal secre- phocytes that have cytotoxic activity independent of tions from mammary glands of dairy cows infected MHC, utilizing their Fc receptors to participate in with Staphylococcus aureus revealed a subpopula- antibody-dependent, cell-mediated cytotoxicity. The tion of activated CD8+ lymphocytes that are capable NK cell binds tumor or virus-infected cells and de- of altering or suppressing the proliferative responses granulates to secrete perforin that destroys the target of CD4+ lymphocytes (38). The immunoregulatory cell by membrane disruption. NK cells also are capa- roles of CD8+ lymphocytes also greatly depend on ble of killing both gram-positive and gram-negative lactation stage. Cells obtained from mid-lactation bacteria, and therefore could be important in pre- dairy cattle exhibited cytotoxic activity and mainly venting mammary infection (44). Changes in this cell expressed IFN-γ , while CD8+ lymphocytes obtained population during the periparturient period have not during the postpartum period exhibited no cytotoxic been studied extensively, but the potent bactericidal activity and mainly expressed IL-4 (39). These data activity of these cells makes them worthy of future suggest that a preferential trafﬁcking of CD8+ sup- study. pressor lymphocytes into mammary gland tissues and secretions may be responsible for the lower respon- siveness of local lymphocyte populations compared Soluble Defenses with those from later stages of lactation. The γ δ T cells are not as well characterized, but Both speciﬁc and innate soluble factors repre- data have suggested that they can be cytotoxic and sent an important line of defense within the mam- may provide a unique line of defense against bacte- mary gland that can elicit effective protective re- rial infections. The γ δ T lymphocytes preferentially sponses to invading pathogens. The primary soluble migrate to epithelial surfaces and do not circulate ex- effectors of the speciﬁc immune response are anti- tensively (40). There are indications that γ δ T lympho- bodies produced by antigen-activated B lymphocytes. cytes, like natural killer cells, can mediate cytotoxicity There are four classes of Ig known to inﬂuence the 140 Sordillo and Streicher Table II. Summary of Mammary Gland Soluble Defenses production proﬁle is somewhat similar in sows, as there is a reduced number of IgA-bearing cells in Factor Biological function mammary secretions at parturition (21). In humans, Cytokines Proinﬂammatory and immunoregulatory IgA secretion was shown to be signiﬁcantly reduced in factors the normal milk of women who ultimately developed Complement Bacteriolytic and/or facilitates mastitis, compared to women who remained mastitis- phagocytosis Lysozyme Cleaves carbon bonds and disrupts free (9,47). During the periparturient period, changes bacterial cell walls in the levels and activity of these Ig isotypes contribute Lactoferrin Sequesters iron to prevent bacterial to the generalized immunosuppression that leads to uptake; disrupts bacterial cell wall; increased incidence of disease. regulates mammary leukocyte activity In addition to the speciﬁc effects of antibodies, Antibodies IgG1 Selectively transported into mammary the mammary gland has nonspeciﬁc bacteriostatic fac- secretions; opsonizes bacteria to tors that work together with or independent of Ig. One enhance phagocytosis such factor is lactoferrin, an iron-binding protein pro- IgG2 Transported into secretions during duced by epithelial cells and leukocytes that functions neutrophil diapedesis; opsonizes to bind free ferric ions in milk, thereby preventing the bacteria to enhance phagocytosis IgA Associated with the fat portion of milk; growth of bacteria that need iron for growth (48,49). does not bind complement or opsonize In ruminants, lactoferrin and IgG1 act synergistically particles; can cause agglutination, to inhibit Escherichia coli and Klebsiella pneumoniae, prevent bacterial colonization, and but other bacteria like Streptococcus agalactiae can neutralize toxin actually use lactoferrin as an iron source. In the lactat- IgM Efﬁcient at complement ﬁxation, opsonization, agglutination and toxin ing bovine mammary gland, the lactoferrin concentra- neutralization; only opsonic for tion is lower than that seen during involution and in- neutrophils in the presence of ﬂammation. In addition, the bacteriostatic activities of complement lactoferrin can be abolished in the presence of citrate, a buffer produced by epithelial cells which chelates iron into a form that is readily usable by bacteria (50). bacterial defense mechanisms of the mammary gland: Accordingly, it has been suggested that lactoferrin’s IgG1 , IgG2 , IgA, and IgM (45) (see Table II). In gen- main role in mammary gland defense is to protect eral, Ig reaches its peak concentrations in mammary against coliform infection, especially during involu- secretions during colostrogenesis and during inﬂam- tion. In humans, studies have identiﬁed lactoferrin as mation. IgG1 is the primary isotype found in healthy a predisposing factor for mastitis by showing that low mammary secretions, but IgG2 increases substantially lactoferrin levels in normal milk correlate with the during mammary gland inﬂammation. Several iso- subsequent development of mastitis, and that lactofer- types (IgG1 , IgG2 , and IgM) can act as opsonins to en- rin deﬁciency contributes to an increased propensity hance phagocytosis by neutrophils and macrophages. of recurring mastitic infections (51). On the contrary, IgA does not aide in bacterial op- Complement is a collection of proteins present sonization, but functions instead in the agglutination in serum and milk that can impact both innate and of invading bacteria that can prevent the spread of acquired immunity. The proteins that comprise the bacterial disease in the mammary gland. Previous re- complement system are synthesized mainly by hepa- search has shown that the concentration of IgG in tocytes, but other sources include monocytes and tis- bovine serum is lower at parturition and suggested sue macrophages. Many of the biological activities of that the lack of the IgG2 isotype speciﬁcally correlates complement are mediated through complement re- to an increased incidence of mastitis (6). Interest- ceptors located on a variety of cells. Effector functions ingly, a lower incidence of mastitis has been corre- of complement include lysis of bacteria, opsonization, lated with high antibody responsiveness to ovalbumin and the attraction of phagocytes to the site of comple- in dairy cows (46). However, this study also showed ment activation. For example, gram-negative mastitis- that not all dairy cows experience a reduction in an- causing bacteria such as Escherichia coli are especially tibody responsiveness at parturition, implying that sensitive to complement-mediated lysis. Complement some animals may not develop disease due to a supe- also functions in concert with a speciﬁc antibody rior, natural ability to produce high levels of antibody as an opsonin which will promote bacterial phago- regardless of the lactation stage (46). The antibody cytosis and intracellular killing by mammary gland Immunobiology of the Mammary Gland 141 neutrophils and macrophages (52). Complement con- of this antimicrobial system against the pathogens that centrations in mammary gland secretions have been cause mastitis. determined by measuring hemolytic and bactericidal Numerous reports have shown the immunomod- activities (53,54). The lowest concentrations of com- ulatory capabilities of cytokines on important mam- plement are observed in the milk of healthy mammary mary leukocyte functions (56–58). The major groups glands during lactation. In contrast, the highest con- of cytokines studied to date include interleukins (IL), centrations of complement are observed in colostrum, colony-stimulating factors (CSF), interferons (IFN), mastitic milk, and in mammary secretions obtained and tumor necrosis factors (TNF) (see Table III). during involution, presumably due to the mobiliza- The term “interleukin” was originally introduced tion of complement components by transudation from to describe cell-free soluble factors that function blood (54). While the overall signiﬁcance of the com- plement system in mammary gland defense has yet to be fully deﬁned, available information does suggest Table III. Cytokine Effects on Mammary Immune and Inﬂamm- atory Responses a predominant role as a proinﬂammatory mediator during coliform mastitis (53). Cytokine Observations Lysozyme is a bactericidal protein that is present in milk and functions by cleaving peptidoglycans from IL-1 Mediates acute phase inﬂammatory response the cell wall of Gram-positive bacteria, as well as Increases neutrophil numbers from the outer membrane of Gram-negative bacte- Enhances neutrophil phagocytosis and bactericidal activity ria. Lysozyme may enhance the binding of lactoferrin Triggers neutrophil migration into infected to bacterial cell walls. In porcine and human milks, mammary gland lysozyme, in combination with complement and se- IL-2 Enhances mammary mononuclear cell cretory IgA, exhibits signiﬁcant bactericidal activity proliferation to E. coli in vitro (55). Because ruminant milk con- Enhances cytotoxic and bactericidal activities of lymphocytes tains only a small concentration of IgA and consid- Increases plasma cell numbers erably less lysozyme than human milk, this system Activates NK cells may offer little protection to the bovine mammary IL-8 Induces inﬂammation gland. Mediates IL-1 induced neutrophil migration The enzyme lactoperoxidase, in the presence of Potent chemoattractant G-CSF Increases numbers of blood and milk thiocyanate and hydrogen peroxide, is bacteriostatic neutrophils for Gram-positive and Gram-negative bacteria. How- Increases milk SCC ever, several factors can vary the effectiveness of this Increases phagocytosis and bactericidal system in mammary gland epithelial cells. Lactoper- activity oxidase is produced in small concentrations by the Decreases neutrophil migration GM-CSF Enhances chemotactic and bactericidal mammary gland. The levels of thiocyanate in the activities of neutrophils mammary gland are dependent on the speciﬁc di- Enhances cytotoxic activity etary composition of the cow. The hydrogen perox- Increases number of phagocytic cells ide in the mammary gland is generated by enzymatic M-CSF Regulates proliferation and differentiation constituents of milk and, if present, by Streptococci. of macrophages Potent macrophage chemoattractant The lactoperoxidase-thiocyanate-hydrogen peroxide IFN-γ Enhances neutrophil phagocytosis and system exerts its antibacterial properties through the bactericidal activity production of hypothiocyanate, a reactive metabo- Reverses suppressive effects of mammary lite formed from the oxidation of thiocyanate (55). gland secretions Myeloperoxidase produced by neutrophils catalyzes TNF-α Enhances acute phase inﬂammatory response the same peroxidase reaction as lactoperoxidase and Enhances neutrophil phagocytosis and additionally catalyzes the oxidation of chloride, the bactericidal activity product of which provides the bactericidal activity of Enhances endothelial adhesion molecule this system. In humans, myeloperoxidase is entirely expression responsible for the catalytic activity of this antimicro- Note. IL = interleukin, G-CSF = granulocyte colony-stimulating bial system in milk (55). However, the low oxygen ten- factor, GM-CSF = granulocyte-monocyte colony-stimulating fac- sion of the mammary gland can inhibit the production tor, M-CSF = macrophage colony-stimulating factor, IFN-γ = of hydrogen peroxide, thus limiting the effectiveness interferon-γ , and TNF-α = tumor necrosis factor-α. 142 Sordillo and Streicher as communicator molecules between leukocytes. Al- granulocytes. Treatment of bovine peripheral blood though all cytokines share this basic property, many and mammary gland neutrophils with rbGM-CSF of the well-characterized cytokines are designated as signiﬁcantly increased the chemotactic and bacterici- IL; IL-2 is the most extensively characterized of all dal capabilities of these cells. Intramammary infusion the bovine cytokines. Originally described as T-cell of rbGM-CSF at doses of up to 5 mg did not signif- growth factor, IL-2 is primarily produced by T lym- icantly affect total milk somatic cell counts (SCC), phocytes of the Th1 phenotype and is responsible for but increased the ability of resident neutrophils to clonal expansion of the initial T lymphocyte immune produce superoxide and increased the percentage of response and establishment of immunologic mem- phagocytic cells (56). Because an early and rapid re- ory following mitogenic or antigenic stimulation. This gression of functionally competent neutrophils from cytokine also plays a role in B lymphocyte growth the blood stream is critical for the control of new in- and differentiation, enhancing thymocyte prolifera- tramammary infections (IMI), the enhancement of tion, activating NK cells, and inducing cytotoxic T-cell chemotaxis and phagocytosis by GM-CSF, as well activation. There is evidence that decreased endoge- as the kinetics of this enhancement, could increase nous IL-2 production contributes to diminished im- the resistance of the mammary gland to invading mune capabilities, which can lead to the development pathogens. of disease. In fact, studies showed that colostrum sam- Interferons are a group of closely related pro- ples that were obtained during the ﬁnal week of ges- teins of two major classes. Class I IFN consists of tation had low IL-2 activity, which correlates with three related types: IFN-α, IFN-β, and IFN-ω. The diminished immune cell function and increased sus- IFN-α and IFN-β are produced by a variety of cell ceptibility to mastitis during this period. The possibil- types in response to several inducers, including viral ity of enhancing bovine mammary gland defenses with infections, bacterial products, and tumor cells. In the IL-2 to increase resistance to mastitis has received bovine, the IFN-ω genes code for proteins produced considerable attention. In vitro and in vivo studies in- by the early embryonic trophoblast, and these are re- dicated that recombinant bovine IL-2 may enhance ferred to as IFN-τ . The second class of IFN consists of the functional capabilities of populations of mononu- a single protein, IFN-γ, which is unrelated to the class clear cells within the mammary gland (55,59). I IFN. Interferon-γ is a cytokine derived from T lym- The CSF are a group of cytokines required for phocytes that is often produced in response to stim- the proliferation and differentiation of a variety of ulation by antigens or mitogens. Evidence suggests hematopoietic stem cells. These growth factors are that IFN-γ could elicit functional changes in phago- distinct glycoproteins that bind to cells by a com- cytic cells in the mammary gland that could make it mon receptor and are produced by a variety of cells, effective in the control of bovine mastitis (57). The including ﬁbroblasts, endothelial cells, macrophages, in vitro treatment of bovine mammary gland neu- and T cells. Each CSF tends to target a speciﬁc cel- trophils with IFN-γ was shown to reverse the sup- lular lineage to expand or activate its function. The pressive effects of mammary gland secretions and sig- pronounced inﬂuence of granulocyte (G)-CSF on niﬁcantly increase the functional capabilities of these phagocytic cell populations suggests possible clini- cells against S. aureus. cal applications in the prevention of infectious bac- The acute symptoms most often associated with terial diseases, such as mastitis. Recombinant hu- coliform mastitis are due to the rapid and unrestricted man G-CSF has been administered subcutaneously growth of the organism and the subsequent devel- to cows in doses ranging from 1 to 5 µg/kg per opment of an unlimited inﬂammatory reaction. Of day, resulting in an increase in peripheral blood neu- the acute phase cytokines produced during the early trophils after 3–5 days of injections (60). Treatment stages of infection, TNF-α is a major factor that causes with G-CSF was shown to decrease random and di- endotoxic shock during peracute coliform mastitis. El- rected neutrophil migration and increase phagocyto- evated sera and milk concentrations of TNF-α were sis and bactericidal activity. Granulocyte-macrophage found in cows that had died from acute E. coli masti- (GM)-CSF was ﬁrst identiﬁed by its capacity to induce tis during the periparturient period (57). Monocytes hematopoietic progenitor cells to develop into gran- isolated from periparturient dairy cows can produce ulocytes and macrophages. Several studies in dairy more TNF-α following LPS stimulation than cells iso- cows have shown that GM-CSF is not only an impor- lated from cows in mid to late lactation (61). The en- tant molecule for inducing the growth of these cell hanced ability of localized cell populations to pro- types, but also affects a variety of functions of mature duce this potent proinﬂammatory mediator around Immunobiology of the Mammary Gland 143 the time of calving may explain the greater frequency sired level. Immunomodulatory strategies may alter of clinical coliform mastitis during the periparturient host defense to evoke a change in the magnitude period. The ability to modify its production during or kinetics of immune or nonimmune responses to coliform mastitis may lessen the morbidity and mor- either a speciﬁc antigen or a range of antigenic stimuli. tality associated with the acute form of this disease. The principle targets of immunomodulation include Stresses due to pregnancy and parturition stim- T and B lymphocytes, NK cells, cells of the mono- ulate the production of a variety of stress hormones cyte/macrophage series, and granulocytes. However, that can have important effects on the immune re- soluble components of the immune system, such as cy- sponse. One group of these stress hormones known tokines, immunoglobulins, and complement also are to cross-regulate immune function is the corticos- amenable to immunomodulatory strategies. Since im- teroids. The synthetic glucocorticoid dexamethasone munoregulatory agents can act on more than one cell can decrease the total number, distribution, and func- type, the ﬁnal biological effect in the animal will de- tion of leukocytes in bovine blood (2). As shown pend on the contribution of the targeted immune com- previously, studies of neutrophils during parturition ponent to various immune responses and the relative revealed a down-regulation of adhesion molecules sensitivity to the immunomodulatory agent. (CD62L and CD18), which correlates to neutrophil The design of immunomodulatory strategies dysfunction and disease development in cattle (62). should consider critical host–pathogen interactions A potential mechanism explaining this effect could involved in the pathogenesis of disease. Mastitis is be that the dexamethasone-induced down-regulation caused by a wide array of bacteria with diverse modes of the glucocorticoid receptor (GR) on neutrophils of pathogenesis. Mastitis-causing pathogens possess alters GR-mediated gene expression of CD62L, lead- a spectrum of virulence factors that facilitate the ing to impaired neutrophil migration during the peri- colonization and infection of the mammary gland. parturient period (62). Additionally, glucocorticoids For example, certain mastitis-causing pathogens can cause a decrease in γ δ T cells in the blood, lower IgM evade host defenses by adhering to epithelial cells, in mammary secretions, reduced expression of MHC producing capsules to prevent ingestion and destruc- on mononuclear cells, and an inhibition of cytokine tion by neutrophils, producing endo- and enterotox- production (24,63–65). ins to destroy or inactivate leukocytes, and utilizing In addition to the effects mediated by the intracellular invasion to escape immune surveillance. corticosteroid family of stress hormones, a number If successful, the bacteria and their by-products not of other stress-associated factors have been shown only exacerbate the inﬂammatory response, but also to be altered at parturition. For example, changes damage the surrounding secretory parenchyma and in progesterone, estradiol 17β, insulin-like growth vasculature. In order to prevent the establishment factor 1 (IGF), and growth hormone (GH) have of disease, the host must respond appropriately to the potential to modify neutrophil and lymphocyte neutralize the impact of these bacterial virulence fac- function (6,66–68). In the porcine system, estradiol tors. The purpose of any immunomodulator is to pro- 17β and cortisol concentrations were decreased mote a more effective and sustained immunity to in- in mammary secretions, which led to virtually no fectious agents without the risk of toxicity or tissue change in the phagocytic capacity of neutrophils, damage. The design of effective immunomodulators but a signiﬁcant decrease in the opsonic activity of for the control of mastitis is especially difﬁcult be- these secretions (28). In humans, psychological stress cause the disease is caused by a wide variety of infec- has been linked to lowered levels of IgA and cited tious agents with distinctly different modes of patho- as a major risk factor for mastitis in breastfeeding genesis. Moreover, the impact of certain bacterial women (9). Although no direct associations have pathogens is determined by stressful environmental been made, the possibility exists for a link between and/or physiological conditions. Therefore, one needs low levels of IgA, increased stress, and an increased to completely understand the pathogenesis of speciﬁc incidence of mastitis. mastitis-causing pathogens to determine if prevention or therapy with an immunomodulator is a viable op- FUTURE PROSPECTS FOR tion. 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