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* Department of Animal Science, McGill University, Ste. Anne de Bellevue, Québec, H9X 3V9, Canada; and and
Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, PO Box 90 STN Lennoxville, Sherbrooke, Quebec, J1M 1Z3, Canada
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Abstract |
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Key Words: bovine • mammary gland • mastitis • epithelial cell • apoptosis • tissue damage
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INTRODUCTION |
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Mastitis is the most costly infectious disease of dairy cattle. The prevalence of mastitis in dairy cattle is relatively high. Subclinical mastitis is the main form of mastitis in modern dairy herds, exceeding 20 to 50% of cows in given herds (Wilson et al., 1997
; Pitkala et al., 2004). The cost of subclinical mastitis is very difficult to quantify, but most experts agree that subclinical mastitis costs the average dairy farmer more than does clinical mastitis. Assuming a 45% prevalence of subclinical mastitis, the cost has been estimated in the range of $180 to $320 per case (Wilson et al., 1997). Approximately 70% of this cost is associated with a reduction in milk production. A large portion of it results from irreversible damage to the mammary tissue (Oliver and Calvinho, 1995). Although antibiotics are very useful to treat the infection, they do not directly protect the gland from being damaged.
This review is a compilation of some major findings concerning tissue damage during mastitis. We describe the current understanding of how bacteria, polymorphonuclear neutrophils (PMN), and host proteases and cytokines contribute to tissue damage. Finally, research aimed at the reduction of tissue damage is discussed.
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OVERVIEW OF MASTITIS |
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). Bacteria may escape the natural defense mechanisms by multiplication along the streak canal (especially after milking), or by propulsion into the teat cistern by vacuum fluctuations at the teat end during milking. The infection occurs after bacteria gain entrance to the mammary gland via the teat canal. After bacteria overcome the anatomical defense, they must evade the cellular and humoral defense mechanisms of the mammary gland to establish disease (Sordillo and Streicher, 2002). If the infection is not eliminated, bacterial levels in the mammary gland eventually rise to a level at which they begin to damage the mammary epithelium. As infection persists, the number of somatic cells in milk continues to increase and, concomitantly, tissue damage is worsened. The alveoli in the gland start to lose structural integrity and the blood-milk barrier is breached. This allows extracellular fluid to enter the gland and mix with the milk. Visible changes in the milk and the udder start to occur. These can include external swelling, reddening of the gland, and clotting and wateriness of the milk. By definition, this is the start of clinical symptoms. In brief, bovine mammary epithelial cells can be damaged during IMI by 1) release of a range of cellular and extracellular products from bacterial pathogens; 2) lysosomal enzymes and oxidative products released from phagocytes during phagocytosis of invading organisms, and 3) proteases from blood and cytokines released during the immune response. |
APOPTOSIS AND NECROSIS |
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). Apoptosis may be identified by a characteristic pattern of morphological changes, including nuclear and cytoplasmic condensation, nuclear fragmentation, and formation of apoptotic bodies (Strange et al., 1992). These changes are associated with cleavage of chromatin into discretely sized oligonucleosome fragments by a calcium-dependent endonuclease (Arends et al., 1990). Appearance of this oligonucleosomal DNA laddering in stained gels is now widely used to detect apoptosis. Enzymatic labeling of DNA strand breaks also allows for histological detection of apoptosis in the tissue by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. In addition, loss of asymmetry in cell membrane phospholipids during apoptosis allows Annexin-V staining to be used to detect exposed phosphatidylserine as a marker for apoptosis. Furthermore, specific caspases, specific factors in apoptotic pathways, and cleavage of caspase substrates have been explored to detect apoptosis. Unlike apoptosis, necrosis refers to the accidental death of cells in living tissue. Necrosis may follow a wide variety of injuries, both physical (e.g., cuts, burns, bruises) and biological (e.g., effects of disease-causing agents). The sign of necrosis, dead tissue, is called a lesion. It begins with swelling of the cytoplasm and mitochondria caused by a loss of membrane integrity and ends with total cell lysis.
These apparently clear-cut definitions between apoptosis and necrosis, however, are likely oversimplified. For example, the apoptotic bodies as well as the remaining cell fragments, if not quickly removed by phagocytes or neighboring cells, will ultimately swell and finally lyse. This process is called secondary necrosis. In addition, the positive or negative interconnection between autophagy and apoptosis (Gozuacik and Kimchi, 2004) adds additional complexity to apoptosis research.
Cytotoxicity is also commonly used in the literature to describe tissue damage, but does not infer a specific cellular mechanism. Rather, it indicates the cell-killing property of a chemical compound or a mediator cell. Many assays are available for measuring cytotoxicity, and most of them are based on increased plasma membrane permeability or increased uptake of dyes by dying cells or dead cells. The results from cytotoxicity assays vary considerably depending on the method used (Fellows and O’Donovan 2007). In general, cytotoxicity assays can be used to detect necrosis and the late stage of apoptosis, but not the early stage of apoptosis. Specifically for bovine mammary studies, lactate dehydrogenase activity and N-acetyl-β-D-glucosaminidase (NAGase) activity have commonly been used as markers for tissue damage. The former enzyme is a stable cytoplasmic enzyme present in all cells. It is rapidly released outside the cell when the plasma membrane is damaged. Located within lysosomes of mammary epithelial cells, NAGase is released from damaged mammary epithelial cells (Kitchen et al., 1980). However, a small fraction of milk NAGase can also come from leukocytes (Capuco et al., 1986).
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TISSUE DAMAGE: HISTOLOGICAL OBSERVATION |
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examined the mammary parenchyma from 184 slaughtered dairy cows for the existence of microorganisms and histopathological changes. Of all the samples from which microorganisms were isolated, only 3.1% did not show histological changes. The remaining 96.9% of samples showed an inflammatory response (i.e., edema, mammary epithelial cell damage, and PMN infiltration), tissue repair process, or both. In contrast, 33.9% of samples from glands without evidence of microorganisms (n = 56) showed no histological changes. These results clearly indicate that the presence of microorganisms is associated with tissue damage.
Escherichia coli is one of the most important pathogens causing mastitis in dairy cows (Bradley, 2002), resulting in inflammation that ranges from subacute to peracute. Necrosis of the mammary epithelium occurs during severe, naturally occurring clinical E. coli mastitis, as well as during severe experimental E. coli mastitis. In moderate cases of E. coli mastitis, there is minimal alveolar tissue damage, as shown by Frost et al. (1980). In that experiment, the main changes were superficial and confined to the epithelium of the teat sinus, lactiferous sinus, and large ducts, without serious involvement of the secretory tissue. There were some lesions of the basement membrane underlying the damaged epithelium but damage was localized. In very severe cases, the infection progressed via the ductile system to produce a limited inflammatory reaction but with an extensive involvement of the secretory tissue (Frost and Brooker, 1986). In its most severe form with uncontrolled bacterial multiplication, all lactiferous sinus epithelia were lost, interstitial tissue became hemorrhagic, and often the animal died of toxemia within a few hours of infection (Burvenich et al., 2003). The severity of the disease varies considerably from cow to cow. Kornalijnslijper et al. (2004) reported that 6 h after intramammary inoculation, the bacterial counts, which were significantly correlated with severity of the disease, varied considerably (i.e., up to 800 fold). They concluded that bacterial growth in the phase before massive influx of PMN is a major determinant in the final severity of experimental E. coli mastitis. To determine whether the endotoxin lipopolysaccharide (LPS) is responsible for E. coli-mediated tissue damage, Capuco et al. (1985) cultured lactating mammary tissue explants in the presence of E. coli endotoxin and a laboratory culture filtrate of E. coli. Results indicated that endotoxin had no effect, but the culture filtrate damaged tissue and decreased milk synthesis and secretion.
One of the most common types of chronic mastitis is caused by Staph. aureus. Histopathological responses of lactating tissue to experimental or naturally occurring Staph. aureus mastitis were extensively studied during the 1970s and 1980s. Chandler and Reid (1973) examined mammary parenchymal tissue samples from lactating cows infected naturally with Staph. aureus and reported a massive PMN infiltration and necrosis of secretory tissues. In addition, Heald (1979) observed that mammary tissues from lactating cows inoculated with Staph. aureus exhibited less milk synthesis and secretion, as evidenced by more interalveolar stroma and involuting alveolar epithelium and less alveolar luminal space compared with uninfected contralateral controls. Moreover, these changes were associated with replacement of secretory tissue with nonsecretory tissue (Nickerson and Heald, 1981). Similarly, a study with dry cows revealed that quarters with intramammary Staph. aureus infection had a greater degree of mammary involution with a greater proportion of interalveolar stroma and a lesser proportion of alveolar lumen compared with uninfected quarters (Sordillo and Nickerson, 1988). Finally, a challenge study with heifers by Trinidad et al. (1990) showed that mammary glands infected by Staph. aureus also exhibited fewer alveolar epithelial and luminal areas but more interalveolar stroma than controls. From these results, we are relatively confident in concluding that Staph. aureus infection causes necrosis of the secretory tissues and that the damaged secretory tissue is replaced with non-secretory tissue.
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APOPTOSIS DURING MASTITIS |
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). Apoptosis is a key feature of mammary gland development and function. In addition, apoptosis is critical for removing the milk-secreting alveolar epithelial cells during lactation and postlactational involution (Capuco and Akers, 1999; Capuco et al., 2003). However, conclusive and direct evidence for involvement of apoptosis during mastitis has been provided only by Long et al. (2001). Escherichia coli-infected mammary glands were biopsied, with the resulting tissues processed for RNA, protein, and histological examinations. Both mRNA and protein analyses indicated up-regulation of the proapoptotic factors Bax and IL-1β-converting enzyme, and a down-regulation of the antiapoptotic factor Bcl-2. Further, induction of a 92-kDa gelatinase was observed by gelatin zymography. Finally, the number of apoptotic epithelial cells per 10 microscopic sections, as determined by the TUNEL assay, increased from 1.8 ± 0.5 to 8.8 ± 2.8 cells. In addition, there was evidence for induction of apoptosis by other mastitis pathogens. For example, Sheffield (1997) reported a 5-fold induction of a putative marker of apoptosis, testosterone-repressed prostate mucin-2 mRNA, after experimental infection of the bovine mammary gland with Strep. agalactiae. Finally, in vitro studies indicated that Staph. aureus caused apoptosis in a bovine mammary cell line (Bayles et al., 1998). Nevertheless, the effect of Staph. aureus and Strep. agalactiae on apoptosis of mammary tissue still needs to be confirmed in vivo. It is worthwhile to indicate that apoptosis does not cause major histomorphological changes, as observed in traditional mastitis studies; however, it does reduce cell numbers in mammary tissue. In addition, apoptosis may cause tissue damage in a delayed fashion through secondary necrosis (Medan et al., 2002).
The clearance processes of apoptotic cells during mastitis are not totally understood. The clearance of apoptotic cells by phagocytes, such as macrophages, has been shown. In addition, mammary epithelial cells are known to be capable of phagocytosing apoptotic cells in species other than cattle (Monks et al., 2002). Because methods for detection of apoptosis reveal only the cells involved in the process, it is quite possible that apoptosis during mastitis is underestimated.
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INVOLVEMENT OF NEUTROPHILS IN TISSUE DAMAGE |
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; Riollet et al., 2000a). These PMN are considered as the second line of defense of the mammary gland. The presence of functional PMN is crucial to the host defense against bacterial pathogens (Paape et al., 2003).
The main functions of PMN are to engulf pathogens and destroy them via oxygen-dependent and oxygen-independent systems. At the same time, PMN can potentially harm the mammary gland. The exact mechanism by which PMN damage bovine epithelial cells during mastitis is still not fully understood. Neutrophils may promote tissue injury and disturb mammary function, via reactive oxygen metabolite generation (i.e., the respiratory burst) and granular enzyme release (i.e., degranulation; Paape et al., 2002).
During experimentally induced Staph. aureus mastitis, migration of PMN through the secretory epithelium was correlated with extensive morphological damage (Harmon and Heald, 1982). Direct evidence that PMN damage mammary tissue was provided by Capuco et al. (1986). Neutrophils isolated from mammary glands of nulliparous heifers given an injection of E. coli endotoxin were incubated with mammary tissues from non-infected quarters. Microscopic examination indicated that epithelial cell damage resulted from treatment with intact, lysed, or phagocytosing PMN. The greatest morphological damage resulted from treatment with phagocytosing PMN. This observation was confirmed in an in vitro coculture system with mammary epithelial cells and PMN. Activated blood PMN were cytotoxic for mammary epithelial cells (Ledbetter et al., 2001; Lauzon et al., 2005), possibly via the release of extracellular reactive oxygen species, such as hydroxyl radicals (Boulanger et al., 2002). Oxidative stress can damage all types of biomolecules (e.g., DNA, proteins, lipids, and carbohydrates) and therefore induce tissue injury.
Bovine PMN have primary (azurophilic), secondary, and tertiary granules (Paape et al., 2002, 2003). These intracellular granules contain bactericidal peptides, proteins, and enzymes such as elastase, other proteinases, and myeloperoxidase that are released into phagocytic vacuoles or the extracellular environment (Faurschou and Borregaard, 2003). Proteolytic enzymes in PMN include neutral and acidic proteases. Elastase (EC 3.4.21.36) and cathepsin G (EC 3.4.21.20) are the predominant enzymes in PMN (Bank and Ansorge, 2001). Other proteases in PMN include the thiol protease cathepsin B (EC 3.4.22.1) and the acid protease cathepsin D (EC 3.4.23.5), as reported by Owen and Campbell (1999). Furthermore, activated bovine PMN can express matrix metalloproteinase (MMP)-9 (Li et al., 1999).
Milk PMN usually have lower total proteolytic enzymatic activities than peripheral blood PMN (Prin-Mathieu et al., 2002), indicating that PMN release many proteases during migration into the mammary gland. Using an E. coli mastitis model, Haddadi et al. (2006) observed that milk PMN had lower cathepsin and collagenase activities than peripheral blood PMN. Thus, PMN appear to use up part of these enzymes during their migration to cross the endothelium, extracellular matrix (ECM), and epithelium (Faurschou and Borregaard, 2003). In contrast, Le Roux et al. (2003) reported that elastase was higher in milk than in blood. Elastase is localized in azurophilic granules of PMN, and its increase in milk is probably due to proinflammatory cytokines such as IL-6, IL-2, and tumor necrosis factor-
, which increase the transcription of serine proteases, including elastase and cathepsin G (Le Roux et al., 2003). Thus, the amount and variety of enzymes released by PMN into milk seem to be affected by the migration and activation of PMN.
The enzymes involved in bovine mammary tissue destruction were investigated by Mehrzad et al. (2005) by using an endotoxin-induced mastitis model. They reported that mastitic milk proteases hydrolyzed casein, gelatin, collagen, hemoglobin, mammary gland membrane proteins, and lactoferrin, indicating that mastitic milk proteases have a broad spectrum of activities. Further, the direct involvement of proteases in epithelial cell damage was demonstrated by the fact that coincubation of normal mammary tissue with mastitic milk, but not normal milk, caused tissue degradation (Mehrzad et al., 2005). Therefore, proteases released by PMN are likely involved in mammary tissue damage during mastitis.
It is unlikely that the diapedesis process itself is directly involved in epithelial damage. Under normal conditions, PMN can migrate into mammary glands without damaging the tissue. Further, PMN diapedesis itself did not cause detectable epithelial cell damage in an in vitro study (Lin et al., 1995). However, prolonged diapedesis of leukocytes could cause damage to mammary parenchymal tissue, resulting in decreased production of milk (Harmon and Heald, 1982; Sordillo and Nickerson, 1988). This damage may occur through several mechanisms, including premature activation during migration, extracellular release of toxic products during the killing of some microbes, removal of infected or damaged host cells and debris as a first step in tissue remodeling, or failure to terminate acute inflammatory responses.
The PMN-induced damage is probably somewhat contained. After ingestion and release of their chemicals, most of the milk PMN perish by induction of apoptosis. This is followed by the migration of macrophages into the mammary gland and engulfment of PMN by these macrophages. Through this process, damaging chemicals are walled off within dying PMN, which are then ingested by macrophages to minimize further damage to tissue (Paape et al., 2002, 2003).
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INVOLVEMENT OF BACTERIA IN TISSUE DAMAGE |
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). Much progress has been made in understanding the role of apoptosis and necrosis in response to bacterial infection.
Escherichia coli produces a number of proteinases, including collagenolytic enzymes, which contribute to the degradation of ECM components (Haddadi et al., 2005, 2006). There is no doubt that these proteolytic activities contribute to the apoptosis and necrosis of mammary epithelial cells. However, the degree to which they contribute is still unknown.
Endotoxin LPS is considered a key factor for the pathogenicity of gram-negative bacteria. Accordingly, intramammary infusion of E. coli LPS is often used to study events occurring during E. coli mastitis, because it mimics the symptoms of naturally occurring mastitis without microorganism invasion and toxin production, which could cause direct damaging effects on the mammary epithelial cells (Oliver and Calvinho, 1995). When LPS was used to induce an inflammatory response in the mammary gland, no lesions were observed in the mammary glands (Hill, 1991). Similarly, LPS does not cause tissue damage in vitro in explants of lactating bovine mammary tissue (Capuco et al., 1985) or mammary epithelial cells (Boulanger et al., 2002). However, the possibility for involvement of LPS in E. coli-induced cell damage cannot be totally ruled out, because LPS stimulated the expression of IL-1 by MAC-T cells, an established bovine mammary epithelial cell line (Boudjellab et al., 2000). In addition, LPS increased expression of urokinase-type plasminogen activator (uPA; Ohta et al., 2000). Therefore, it is plausible that LPS induces apoptosis or necrosis in mammary epithelial cells indirectly through induction of proteases or proinflammatory cytokines. At this time, however, there is no experimental evidence to support this notion, presumably because of the previous lack of sensitive analytical methods.
Staphylococcus aureus produces toxins that destroy cell membranes, directly damage milk-producing tissue, and induce necrosis in bovine mammary glands (Chandler and Reid, 1973; Heald, 1979; Nickerson and Heald, 1981; Sordillo and Nickerson, 1988; Trinidad et al., 1990). Initially, the bacteria damage tissues lining the teat and gland cisterns within the quarter. If unchecked, they invade the duct system and establish deep-seated pockets of infection in the milk-secreting cells (i.e., alveoli). This is followed by the walling-off of bacteria by scar tissue and the formation of abscesses. Moreover, Bayles et al. (1998) provided evidence that Staph. aureus induced apoptosis in bovine mammary gland epithelial cells. They showed that 2 h after the internalization of one Staph. aureus mastitis isolate, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolation of the cytoplasm, all of which are indicative of cells undergoing apoptosis. By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the existence of a characteristic DNA ladder pattern and the positive TU-NEL labeling of apoptotic cells. These results clearly demonstrate that after internalization, Staph. aureus escapes the endosome and induces apoptosis in mammary epithelial cells. The apoptosis depends on factors regulated by the global virulence gene regulators Agr and Sar (Wesson et al., 1998), as well as by caspases 8 and 3 (Wesson et al., 2000). Although there is still no direct evidence showing that Staph. aureus induces apoptosis of bovine mammary epithelial cells in vivo, this seems likely.
Staphylococcus aureus produces a wide variety of exoproteins that contribute to its ability to colonize and cause disease in mammalian hosts. Nearly all strains examined secrete a group of enzymes and cytotoxins, which includes 4 hemolysins (, β, 
, and 
), nucleases, proteases, lipases, hyaluronidase, and collagenase. The main function of these proteins may be to convert local host tissues into the nutrients required for bacterial growth. Some strains produce one or more additional exoproteins, which include toxic shock syndrome toxin-1, staphylococcal enterotoxins, exfoliative toxins, and leukocidin (Dinges et al., 2000). Strains isolated from cases of bovine mastitis express , β, , and toxins, leukocidins, enterotoxin, and coagulase (Bramley et al., 1989; Matsunaga et al., 1993). Among all the Staph. aureus cytotoxins, -hemolysin has been the most carefully examined. It can induce both apoptosis and necrosis in eukaryotic cells, depending on the dosage given (reviewed by Weinrauch and Zychlinsky, 1999). At low doses, the toxin binds to specific, but as yet unidentified, cell surface receptors and produces small pores that selectively facilitate the release of monovalent ions, resulting in characteristic DNA fragmentation and cell death. At high doses, -hemolysin nonspecifically absorbs to the lipid bilayer and forms larger pores in the membrane that are Ca2+ permissive, which results in massive necrosis without DNA fragmentation (Weinrauch and Zychlinsky, 1999). Whether this also applies to bovine mammary epithelial cells is unknown. Other bacterial toxins could also have damaging effects on bovine mammary tissues, but they have not been studied intensively. Kuroishi et al. (2003) inoculated mammary glands with staphylococcal enterotoxin C and observed epithelial cellular degeneration, including invagination and cytoplasmic vacuolation of epithelial cells. It is not clear whether this effect is due to increased production of the superoxide by migrated PMN or whether it is a direct effect of staphylococcal enterotoxin C on mammary epithelial cells.
Streptococcus dysgalactiae is an environmental pathogen that causes bovine IMI. Almeida and Oliver (1995) assayed 2 mastitis strains of Strep. dysgalactiae for their ability to invade, multiply, and induce damage to MAC-T cells. They demonstrated that the invasion process did not appear to affect the viability of mammary epithelial cells but that cellular damage was induced, as indicated by the release of increasing amounts of lactate dehydrogenase (Almeida and Oliver, 1995).
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INVOLVEMENT OF PLASMA PROTEINS AND CYTOKINES IN TISSUE DAMAGE |
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). Milk contains 2 proteinase systems derived from blood, one of which is involved in dissolving blood clots (i.e., plasmin) and the other in defense against invasive microorganisms (i.e., lysosomal proteinases of somatic cells). Whereas plasmin is the principal proteinase in good-quality milk, other proteinases, including cathepsins and elastase, are probably also active, particularly as the somatic cell count of milk increases (Kelly et al., 2006; Leitner et al., 2006). This is supported by the observation that the protease activities of plasmin and mastitic milk differ (Mehrzad et al., 2005). In addition, mammary epithelial cells also express certain MMP and serine proteases, which are involved in the activation of plasminogen to plasmin (Green and Lund, 2005).
The plasmin-plasminogen activator system in bovine milk is closely correlated with gradual involution (Politis, 1996). Plasmin directly degrades matrix proteins such as fibrin and laminin and also activates MMP precursors, such as pro-MMP-3, MMP-9, and MMP-13 (Green and Lund, 2005). The increase in plasmin activity during mastitis is linked to the permeability of the milk epithelial barrier during inflammation. In addition, numerous activators of plasmin and its zymogen come from the bloodstream, PMN, and bacteria. During mastitis, the serine protease activator concentration, including those for plasmin and plasminogen in blood and also in milk, increases sharply (Le Roux et al., 2003; Moussaoui et al., 2004). Polymorphonuclear neutrophils have a pool of plasminogen activators, such as uPA and, to a lesser extent, tissue plasminogen activators (Moir et al., 2001). Several bacteria, such as Staph. aureus, E. coli, and Salmonella typhimurium, express a plasminogen receptor on their surfaces, which, through immobilization of plasminogen, enhances plasminogen activation into plasmin (Lahteenmaki et al., 2001).
How much plasmin contributes to tissue damage during mastitis is debatable. Mammary epithelial cell viability depends on attachment to the ECM, so it is reasonable to postulate that ECM degradation is involved in tissue damage and cell death during mastitis. Expression of MMP-9, stromelysin-1 mRNA, and uPA mRNA was increased in association with apoptosis during E. coli mastitis (Long et al., 2001). Matrix metallo-protease-9 can be produced by bovine PMN (Li et al., 1999) and MAC-T cells (Long et al., 2001). Stromelysin-1 contributed to the breakdown of most ECM components, including laminin and collagen type IV (Rudolph-Owen and Matrisian, 1998). Other proteases that have been reported to increase in milk from mastitic cows include MMP-2 and MMP-9 (Raulo et al., 2002; Lauzon et al., 2006), as well as a 120-kDa gelatinase (Mehrzad et al., 2005; Lauzon et al., 2006). Mehrzad et al. (2005) also tested proteolytic activity directly in normal mammary tissue of mastitic cows. They found that lactoserum from mastitic cows was more proteolytic than normal lactoserum. Lactoserum from mastitic cows exfoliated the cells and surrounding proteins, leaving a nude dense collagen network. These differences resulted from protease contents and activities, which were significantly higher in mastitic milk (Mehrzad et al., 2005). They concluded that the proteolysis and tissue damage observed were largely due to MMP. However, the possible contribution from other nonplasmin proteases and plasmin should not be ignored.
Cytokines are central mediators of inflammatory events during IMI. Bacteria release potent toxins that trigger white blood cells and epithelial cells in the mammary gland to secrete cytokines (Paape et al., 2003). Intramammary challenge with E. coli or Staph. aureus has elicited differential innate immune responses in terms of clinical symptoms and milk cytokine profiles at the protein level (Bannerman et al., 2004; Riollet et al., 2000b) and at mRNA levels (Lee et al., 2006). The difference in cytokine profiles may underlie the differences in the sequela of symptoms for these 2 types of mastitis.
Very little work has been conducted to determine the role of cytokines in the regulation of tissue damage during mastitis. Cytokines recruit PMN that function as phagocytes at the site of infection. During a study on the use of recombinant bovine (rb) IL-8 as a potential therapeutic agent for subclinical mastitis of dairy cows, Takahashi et al. (2005) observed significant increases in milk somatic cell counts and in chemiluminescence activity after intramammary injection of rbIL-8. Although they did not study the tissue damage, the high levels of milk somatic cell counts and free radical production would certainly have led to tissue damage. It has previously been reported that rbIL-8 can induce the migration of PMN through an in vitro model of the endo-epithelial barrier consisting of the bovine aortic endothelial cell line and MAC-T cells (Lee and Zhao, 2000). Other cytokines, such as tumor necrosis factor- and IL-1, induce apoptosis in a variety of cell types, including bovine endothelial cells (Mebmer et al., 1999) and human mammary epithelial cells (Burow et al., 1999). Levels of these cytokines increase during E. coli mastitis (Bannerman et al., 2004), and it is tempting to assume that they also induce apoptosis in bovine mammary epithelial cells. A range of cytokines are also known to promote a wide variety of functions of PMN, including adhesion, surface receptor expression, free radical production, and release of lysosomal constituents (Paape et al., 2003). Therefore, the effects of cytokines on tissue damage are more likely to be mediated through recruitment and activation of PMN.
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REDUCTION OF TISSUE DAMAGE |
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).
The major free radicals found in biological systems are superoxide, hydrogen peroxide, hydroxyl radical, and fatty acid radicals (Ward et al., 1988). Hydrogen peroxide is found primarily in the cytosol of cells, and fatty acid radicals are found primarily in cell membranes (Ward et al., 1988). Superoxide and hydroxyl radicals can be found in both cell components (Paape et al., 2003). Because free radicals are extremely toxic to cells, the body has developed a sophisticated antioxidant system. Superoxide dismutase converts superoxide to hydrogen peroxide. Hydrogen peroxide is converted to water by the enzyme glutathione peroxidase (Ward et al., 1988). Those 2 enzymes effectively control most free radicals within the cytosol. Accordingly, the use of antioxidants to prevent oxidative stress has been studied in several types of inflammation (Cuzzocrea et al., 2004).
Using a coculture model of activated bovine PMN and MAC-T cells, Lauzon et al. (2005) found that 3 antioxidants (i.e., catechin, deferoxamine, or glutathione ethyl ester) could partially or totally eliminate the damaging effect of activated bovine PMN to MAC-T cells, indicating that antioxidants may be effective tools for protecting mammary tissue against PMN-induced oxidative stress during bovine mastitis. The protective effects of these 3 antioxidants on PMN-induced damage to mammary cells were further evaluated in vivo by using an endotoxin-induced mastitis model. The extent of cell damage was evaluated by measuring milk levels of lactate dehydrogenase and NAGase activity. Intramammary infusions of catechin or glutathione ethyl ester did not exert any protective effect, whereas infusion of deferoxamine, a chelator of iron, decreased milk lactate dehydrogenase and NAGase activity, indicating a protective effect against PMN-induced damage. The protective effect of deferoxamine was also evident from a lower level of haptoglobin in milk. The proteolytic activity of mastitic milk was not influenced by the presence of deferoxamine. Overall, these results indicate that local infusion of deferoxamine may be an effective tool to protect mammary tissue against PMN-induced oxidative stress during bovine mastitis (Lauzon et al., 2006).
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SUMMARY |
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Footnotes |
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2 Corresponding author: xin.zhao{at}mcgill.ca
Received for publication May 25, 2007. Accepted for publication August 20, 2007.
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LITERATURE CITED |
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-mediated apoptosis in MCF-7 cells. Carcinogenesis 20:2057–2061. and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells. Kidney Int. 55:2322–2337.[CrossRef][Medline]This article has been cited by other articles:
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  Y. H. Schukken, J. Hertl, D. Bar, G. J. Bennett, R. N. Gonzalez, B. J. Rauch, C. Santisteban, H. F. Schulte, L. Tauer, F. L. Welcome, et al. Effects of repeated gram-positive and gram-negative clinical mastitis episodes on milk yield loss in Holstein dairy cows J Dairy Sci, July 1, 2009; 92(7): 3091 - 3105. [Abstract] [Full Text] [PDF]
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Abstract
INTRODUCTION