Taurine attenuates Streptococcus uberis-induced mastitis in rats by increasing T regulatory cells

Taurine (Tau) is reported to have a key role in the regulation of the innate immune response and thus reduce tissue damage induced by bacterial infection. In this study, the effects of Tau on a rat model of mastitis induced by Streptococcus uberis (S. uberis) and the changes of T regulatory cells (Tregs) were assessed. Starting on gestation day 14 and continuing until parturition, 100 mg/kg of taurine (group TS) or an equal volume of physiological saline (group CS) was administered daily, per os. Seventy-two hours after parturition, rats were infused with approximately 100 cfu of S. uberis into each of two mammary glands. The results showed that the resultant inflammation, evidenced by swelling, secretory epithelial cell degeneration, increased adipose tissue and neutrophil (PMN) infiltration were evident in mammary tissue following injection with S. uberis. Pre-treatment with Tau attenuated these morphologic changes, the expression of interleukin (IL)-2, interferon (INF)-γ mRNA, myeloperoxidase (MPO) activity and N-acetyl-β-D-glucosaminidase (NAGase) in mammary tissue. The percentages of Foxp3+CD25+CD4+/lymphocytes (Tregs) were dramatically increased after the S. uberis challenge. Significant differences (P<0.05) were observed at 24, and 72 h post S. uberis-injection (PI) in CS. Pre-treatment further increased the percentage of Tregs and a significant difference between CS and TS (P<0.05) was apparent at 24 h PI. Our data indicate that in rats, Tau can be used to regulate the immune response following infection by S. uberis and consequently prevent mammary tissue damage by increasing Tregs.     

Histone H3K14 and H4K8 hyperacetylation is associated with Escherichia coli-induced mastitis in mice

Mastitis is a multietiological complex disease, defined as inflammation of parenchyma of mammary glands. Bacterial infection is the predominant cause of mastitis, though fungal, viral and mycoplasma infections also have been reported. Based on the severity of the disease, mastitis can be classified into subclinical, clinical and chronic forms. Bacterial pathogens from fresh cow milk were isolated and classified by standard microbiological tests and multiplex PCR. Epidemiological studies have shown that Escherichia coli is the second largest mastitis pathogen after Staphylococcus aureus in India. Based on Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR profile and presence of virulence genes, a field isolate of E. coli was used for intramammary inoculation in lactating mice. Histopathological examination of hematoxylin and eosin stained sections showed severe infiltration of polymorphonuclear neutrophils, mononuclear inflammatory cells in the alveolar lumen and also in interstitial space, and necrosis of alveolar epithelial cells after 24 h. Western blot and immunohistochemical analysis of mice mammary tissues showed significant hyperacetylation at histone H3K14 residue of both mammary epithelial cells and migrated inflammatory cells. Quantitative real-time PCR and genome-wide gene expression profile in E. coli infected mice mammary tissue revealed differential expression of genes related to inflammation, immunity, antimicrobial peptide expression, acute phase response and oxidative stress response. Expression of milk proteins was also suppressed. ChIP assay from paraffinized tissues showed selective enrichment of acetylated histone H3K14 and H4K8 at the promoters of overexpressed genes. These data suggest that E. coli infection in mice mammary tissue leads to histone hyperacetylation at the promoter of immune genes, which is a pre-requisite for the expression of inflammatory genes in order to mount a drastic immune response.     

Activation of nuclear factor kappa B in mammary epithelium promotes milk loss during mammary development and infection

We investigated whether nuclear factor kappa B (NF‐κB), which exhibits a regulated pattern of activity during murine mammary gland development, plays an important role during lactation and involution, when milk production ceases and the gland undergoes apoptosis and re‐modeling. We generated a doxycycline inducible transgenic mouse model to activate NF‐κB specifically in the mammary epithelium through expression of a constitutively active form of IKK2, the upstream kinase in the classical NF‐κB signaling cascade. We found that activation of NF‐κB during involution resulted in a more rapid reduction in milk levels and increased cleavage of caspase‐3, an indicator of apoptosis. We also found that activation of NF‐κB during lactation with no additional involution signals had a similar effect. The observation that NF‐κB is a key regulator of milk production led us to investigate the role of NF‐κB during mastitis, an infection of the mammary gland in which milk loss is observed. Mammary gland injection of E. coli LPS resulted in activation of NF‐κB and milk loss during lactation. This milk loss was decreased by selective inhibition of NF‐κB in mammary epithelium. Together, our data reveal that activation of NF‐κB leads to milk clearance in the lactating mammary gland. Therefore, targeting of NF‐κB signaling may prove therapeutic during mastitis in humans and could be beneficial for the dairy industry, where such infections have a major economic impact. J. Cell. Physiol. 222:73–81, 2010. © 2009 Wiley‐Liss, Inc.     

Endocrine profiles of dairy cows following experimentally induced clinical mastitis during early lactation

Concentrations of LH, cortisol, estradiol-17beta (E(2)), prolactin and 13,14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM) were determined in cows with experimentally induced clinical mastitis during early lactation. Cows free of intramammary infection (IMI) and in the luteal phase of the estrous cycle were balanced by lactation number and days in milk and assigned to either control (n=5) or treatment (n=5) groups. Treated cows were infected experimentally (day 0), in two mammary quarters, with Streptococcus uberis and developed clinical mastitis within 60 h after inoculation as evidenced by increased mastitis scores, elevated rectal temperatures, mammary swelling and isolation of S. uberis pathogen. Four days following bacterial challenge, blood samples were collected every 20 min for 8 h for determination of PGFM and LH following administration of oxytocin and GnRH, respectively. Blood samples were also collected on days 0, 4 and 7 of the experiment to determine concentrations of E(2), prolactin and cortisol. Four days after bacterial challenge, concentrations of cortisol were higher (P=0.04) in experimentally infected cows than controls. Experimentally challenged cows had increased (P=0.02) concentrations of cortisol on days 4 and 7 compared with day 0. Control cows had no significant increase in blood cortisol during the experimental period. Baseline concentrations of PGFM did not differ between groups; however, peak concentrations of PGFM following oxytocin challenge were elevated (P=0.006) in cows with clinical mastitis compared with control animals. Prolactin, E(2) and LH did not differ between cows with clinical mastitis or controls. Experimentally induced mastitis during early lactation elevated concentrations of cortisol during the luteal phase of the estrous cycle. Furthermore, mastitic cows demonstrated an increased PGFM response following oxytocin administration. Altered reproductive efficiency in cows with clinical mastitis caused by Gram-positive pathogens may be the result of increased uterine sensitivity to prostaglandin F(2alpha) (PGF(2alpha)).     

Glucocorticoids maintain the extracellular matrix of differentiated mammary tissue during explant and whole organ culture

Mouse mammary whole organ culture (WOC) and explant culture of lactating tissue were used to investigate the mechanism by which glucocorticoids maintain secretory epithelium following lobuloalveolar development. The relative number of mammary epithelial cells expressing glucocorticoid receptors did not change with the loss of secretory epithelium during involution as demonstrated with competitive binding assays and immunohistochemistry for the glucocorticoid receptor. Furthermore, glucocorticoids did not inhibit AP-1 binding activity. However, Northern analysis demonstrated that genes associated with the breakdown of the extracellular matrix were not expressed in tissues cultured with glucocorticoids, in contrast to their upregulation during involution of mammary tissue cultured with insulin alone. Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression was lowest in tissue cultured in the presence of glucocorticoids and increased 2.3-, 3.4-, and 9-fold when tissues were involuted in the presence of insulin (Ins) alone, Ins and hydrocortisone (Hyd) with 0. 005 mg/ml, or 0.01 mg/ml collagenase IV, respectively. These data indicate that glucocorticoids maintain mammary differentiation in part by inhibiting the turnover of basement membrane.     

INVOLUTION OF MOUSE MAMMARY GLANDS DURING WHOLE ORGAN CULTURE OCCURS VIA APOPTOSIS OF EPITHELIAL TISSUE

Apoptosis was measured in mammary glands during whole organ culture, to determine whether regression resulting from hormone withdrawal results in epithelial cell death as in vivo involution. Glands were evaluated for morphology and DNA degradation prior to whole organ culture, after lobulo-alveolar development and 2, 4, or 6 days after hormone withdrawal. The data indicated that mammary regression during whole organ culture mimics in vivo involution and results in part from apoptosis of epithelial tissue.     

Chronic caprine mastitis in Nigerian goat breeds: microbiological flora and histopathological findings

In a study involving lactating does with normal-sized and enlarged pendulous udders, 85.5% of milk samples obtained from does with unilateral udder enlargement and 84.8% of samples from bilaterally enlarged udders indicated the presence of intramammary inflammation based on results of the modified Whiteside test. In contrast, only 47.6% of milk samples obtained from normal-sized udders showed evidence of intramammary inflammation. Bacterial cultures were performed on all milk samples which showed evidence of intramammary inflammation. 16 species of bacteria and other microorganisms were cultured from these samples. Bilaterally enlarged udders collectively yielded 13 different microbial isolates, while unilaterally enlarged udders yielded 11, and normal-sized udders yielded 9. The most frequent isolates in all groups were Staphylococcus spp. and Corynebacterium spp. On histopathologic examination, severe pathological changes in the teat and udder tissues of enlarged pendulous udders were observed. Lesions in parenchymatous tissues showed mild to severe inflammation with involution of the parenchyma of glands. This led to shrinkage of the alveoli and in severe cases, their complete disappearance and replacement by fibrous connective tissue which was observed to have a high degree of proliferation. Sections of non-pendulous udder tissue showed proper arrangement of alveoli with hypertrophic epithelial cells, indicating active secretory activity. The results provide some definitive evidence that enlarged pendulous udder development in some Nigerian breeds of goats is not spontaneous but occurs largely as a result of incursion of the mammary glands by pathogenic organisms which may cause mastitis resulting in progressive dysfunction of mammary tissue. In chronic cases, there is severe atrophy of glandular tissue leading to fibrosis.     

Vaccination with Staphylococcus aureus fibrinogen binding proteins (FgBPs) reduces colonisation of S. aureus in a mouse mastitis model

Abstract A mouse mastitis model was used to study the effect of vaccination with fibrinogen binding proteins and collagen binding protein from Staphylococcus aureus against challenge infection with S. aureus . The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibronogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. Histopathological examination of tissue sections from challenged glands showed that most glands from mice vaccinated with fibrinogen binding protein developed disseminated necrosis or had no pathological changes. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. In a similar study, immunization of mice with collagen binding protein did not induce protection against challenge infection with S. aureus .     

Persistence of a Staphylococcus aureus small-colony variant under antibiotic pressure in vivo

Staphylococcus aureus small-colony variants (SCVs) have been implicated in chronic and persistent infections. Bovine mastitis induced by S. aureus is an example of an infection difficult to eradicate by conventional antimicrobial therapies. In this study, the ability to colonize mouse mammary glands and persist under antibiotic treatment was assessed for S. aureus Newbould and an isogenic hemB mutant, which exhibited the classical SCV phenotype. The hemB mutant showed a markedly reduced capacity to colonize tissues. However, although the hemB mutant was as susceptible as S. aureus Newbould to cephapirin in vitro, it was over a 100 times more persistent than the parental strain in the mammary glands when 1 or 2 mg kg(-1) doses were administrated. These results suggest that, although the hemB mutant has a reduced ability to colonize mammary glands, the SCV phenotype may account for the persistence of S. aureus under antibiotic pressure in vivo.     

TGF‐β promotes cell death and suppresses lactation during the second stage of mammary involution

Transforming growth factor beta (TGF‐β) ligands are known to regulate virgin mammary development and contribute to initiation of post‐lactation involution. However, the role for TGF‐β during the second phase of mammary involution has not been addressed. Previously, we have used an MMTV‐Cre transgene to delete exon 2 from the Tgfbr2 gene in mammary epithelium, however we observed a gradual loss of TβRII deficient epithelial cells that precluded an accurate study of the role for TGF‐β signaling during involution timepoints. Therefore, in order to determine the role for TGF‐β during the second phase of mammary involution we have now targeted TβRII ablation within mammary epithelium using the WAP‐Cre transgene [TβRII^(WKO)Rosa26R]. Our results demonstrated that TGF‐β regulates commitment to cell death during the second phase of mammary involution. Importantly, at day 3 of mammary involution the Na–Pi type IIb co‐transporter (Npt2b), a selective marker for active lactation in luminal lobular alveolar epithelium, was completely silenced in the WAP‐Cre control and TβRII^(WKO)Rosa26R tissues. However, by day 7 of involution the TβRII^(WKO)Rosa26R tissues had distended lobular alveoli and regained a robust Npt2b signal that was detected at the apical luminal surface. The Npt2b abundance and localization positively correlated with elevated WAP mRNA expression, suggesting that the distended alveoli were the result of an active lactation program rather than residual milk protein and lipid accumulation. In summary, the results suggest that an epithelial cell response to TGF‐β signaling regulates commitment to cell death and suppression of lactation during the second phase of mammary involution. J. Cell. Physiol. 219: 57–68, 2009. © 2008 Wiley‐Liss, Inc.     

Fonction catabolique de l'épithélium mammaire

Summary The histochemical (iron, lipopigments, acid phosphatase, leucine aminopeptidase) and cytologic (lysosomes) changes occuring during pregnancy, lactation and involution of mouse, rat, rabbit, guinea-pig mammary glands are studied by light microscopy and electron microscopy.In all the animals examined, the mammary epithelium has an intracellular digestive system which is adapted to subserve two functions. The first one is the segregation of cytoplasmic components which often precedes cellular involution. The second one is the regulation of secretory processes in the non lactating glands. This digestion of endogenous materials results in the formation of various lytic bodies: dense bodies sometimes containing ferritin, vacuolated dense bodies with membranous residues, autophagic vacuoles. The lysosomes can give large complex dense bodies like lipofuscin pigments with or without ferritin.Leucine aminopeptidase which always disappears in the mouse mammary epithelium during lactation is not present in rat, rabbit, guinea-pig mammary epithelium. In these species only the vascular tissue contains the enzyme. This observation indicates that leucine aminopeptidase does not take care of the overproduction of secretory products in the non-lactating glands.Acid phosphatase is concentrated in secretory granules and in lytic bodies: multivesicular bodies, dense bodies with ferritin, vacuolated dense bodies, lipopigments. This enzyme constitutes probably a mechanism for controlling and triggering the destruction of the secretory material with no active elimination.The iron of the mammary epithelium appears in virgin mice older than 30 weeks and in mice, rats, rabbits, guinea-pigs during glandular cells involution. This is a catabolic iron located in lysosomes. Its amount depends upon the iron content of the milk and upon the competitive secretory and catabolic activities of the glandular cells. An explanation of iron disappearance during a second pregnancy and lactation is discussed.     

Isolation of cell death-associated cDNAs from involuting mouse mammary epithelium

Although apoptosis is important in determining cell fate and maintaining tissue homeostasis, the initiation and control of apoptotic cell death in epithelium is not well understood. Post-lactationai involution of the mammary gland provides both an important developmental process and a normal physiological setting for studying apoptosis of epithelium. We used a differential screening strategy, based on previous studies correlating morphology with gene expression and nucleic acid integrity during mammary gland involution, to isolate genes involved in the regulation and execution of apoptotic cell death in regressing mammary epithelium. This screening strategy yielded a large number of genes the expression of which is significantly altered during mammary gland involution. These include genes associated with cell death processes, tissue remodelling and mesenchymal differentiation. In addition, a number of novel genes have been isolated. We have used Northern analysis and in situ hybridisation to study the expression of a selection of these putative death-associated genes during post-lactational mouse mammary gland involution.     

Enzyme Histochemistry as a Link between Biochemistry and Morphology

A study has been made of the progress of involution of the mouse and rat mammary gland using histologic, electron microscopic, histochemical and autoradiographic methods. Particular emphasis has been placed on the morphology, metabolic alte-rations and activities of histochemically identifiable enzymes, and on the pharmacologic effects of lactation inhibiting agents and cytostatic drugs on lactation and involution. In order to allow a systematic investi-gation, involution was initiated in rats and mice by ligation of individual gland ducts at various time intervals. Both lactating glands and glands in different phases of involution were thus available in a given animal.The most important observation was that involution, which altogether takes approximately 2 weeks to be complete, involves a three-phase process, each phase being clearly distinguishable by morphologic and histochemical criteria. The first phase comprises approximately 4 days during which production of milk may be reinitiated. The second phase starts on day 5 of involution and constitutes the period of involution per se characterized by appreciable parenchymal cell degradation. The third phase, which starts around day 10, is the period of reorganization to the resting mammary gland.Early in the first phase of involution, substantial alveolar enlargement due to engorgement with milk, together with epithelial flattening, are prominent features. By day 3, the glandular contents decrease again in volume, the number of glandular cells and the constituent cytoplasmic organelles remaining unchanged during this period, except for the diminished appearance of fat droplets. In addition to normal appearing vacuoles with only occasional or sparse protein granules, giant vacuoles containing, in part, several hundred casein granules are found. Their formation appears to be due to increased stacking of granules in distended vacuoles prior to dissociation from the Golgi apparatus. In addition, however, the enhanced reactions of a1P (alkaline phosphatase) and ATPase, which are found in the apical plasmalemma, are suggestive of resorptive activities. Protein particles absorbed from the glandular lumen equally appear to have a capacity for fusing into large vacuoles. The large protein granule-containing vacuoles regularly exhibit intense (3-Glu activity. This enzyme would appear to contribute actively to the degradation of excess milk during the first phase of involution.Autoradiographic studies reveal that the synthesis and release of proteins into the secretion is maintained for 3 days. While 3H-tyrosine uptake by the alveolar cells continues unchanged, the incorporation of 3H-palmitic acid into glandular lipoids, and of 3H-fucose into glandular polysaccharides is virtually blocked completely. An immediate reaction of the lipoid metabolism is also indicated by the decrease in 3HBDH activity on the first day of involution.The activities of the histochemically detected oxidoreductases (LDH, MDH, SDH, G6PDH, 3HBDH) show a sharp fall on day 1 of involution, reaching levels approximately one half of the activity observed during lactation, as shown on micro densitometry. The activities remain unchanged during the following 4 days.No degradation of glandular parenchyma is noted during the-first phase of involution. The glandular cells rather take a -wait-and-see- attitude which enables them to participate again in the secretion of milk, as need arises. At this time the activities of the enzymes implicated in energy metabolism have reached approximately the resting mammary gland level. Only protein synthesis is maintained virtually unrestricted and this results in the production of excess milk constituents that are degraded as soon as they are being formed.In the second phase of involution, large seg-ments of the glandular epithelium undergo invo-lution, a process which involves the destruction of glandular epithelial cells and the removal of the resulting cellular debris from the mammary gland. The glandular cells remaining are transformed into resting cells. The lysosomes of the glandular epithelial cells, with maximum numbers being attained between days 7 to 9, contribute decisively to this degradative process. Ultra-structurally, this stage is initially characterized by the appearance in the alveolar cell cytoplasm of segregated cytoplasmic areas which stain negatively for acP (acid phosphatase) and are rich in organelles. These cytoplasmic areas change to membrane-bordered lysosomes which possess intense acP activity. The lysosomes are obviously required for the autophagic degradation of cytoplasmic segments. At the same time the activities of other lysosomal enzymes, involving acP, N-A-Gase, AMPase and AS, show a sharp increase. ATPase and TPPase likewise exhibit considerably increased activity during the second phase of involution. It is seen on microdensitometry that during this phase the acP attains approximately three times the lactation activity. In contrast, the activity of (3-Glu, after having shown a very high increase during the first phase, reverts again to the resting mammary gland level.During the second phase of involution, the oxidoreductases are subject to a further drastic decline of their activities. This process, which consistently affects all segments of the mammary gland,,comes to completion within a few hours. The reaction is found to be minimal around day 5 of involution, at a time when the enzyme activities are approximately one quarter of the lactation levels.At this time, many alveolar cells are destroyed and released into the glandular lumen, the acP retaining its activity in the lysosomes of sloughed cells or cell debris. The resulting gaps in the alveolar epithelium are either bridged immediately or remain detectable on histology. Yet the glandular contents do not pass into the interstitial tissue. The adherence of the glandular tree and the glandular epithelium is ensured by myoepithelial cells. The basement membrane effects the complete segregation of the parenchyma from the interstitial tissue:Macrophages which at this stage occur increa-singly near the alveoli, in the alveolar epithelium and in regional lymph nodes, and which are conspicuous due to numerous acP-laden lysosomes participate essentially in glandular regression.The third phase of involution, which takes place approximately 10 to 14 days following the onset of milk stasis, is the period of reorganization to the resting mammary gland, a period during which glandular cells containing little cytoplasm and sparse organelle lining make their appearance. However, the activities of acP and other lysosomal enzymes remain elevated compared to the pregestational level. Histochemically, the reaction of the oxidoreductases is more intense than during the second involutionary phase, corresponding roughly to the level of the mammary gland in the resting state. The formation of the glandular epithelial cells of the resting mammary gland is not due to renewed mitotic activity, but results from the reduction of actively secreting epithelial cells.Throughout the period of involution the myo-epithelium consistently changes its shape. How-ever, cytoplasmic alterations are not discernible on electron microscopy, nor do these cells undergo degradation during glandular involution. The alP reaction is of particular value for the identification of the myoepithelial cells. No alteration in enzyme activities is demonstrable at the histochemical level throughout the process of involution.Alterations in the interstitial tissue affect particularly the adipose tissue. During lactation, the interalveolar space exhibits only a narrow connective tissue layer which changes insignificantly during the first phase of involution, whereas the subsequent incorporation of lipids results in the formation of plurilocular lipid cells and the reappearance of unilocular adipose tissue as involution advances. During this period the vessels move away from the alveoli.The response of the mammary gland to lactation inhibiting and cytostatic drugs varies, depending on the agent administered. Estrogencontaining drugs lead to an involutionary process which in its initial phase differs from that observed during normal glandular regression. Due to the fact that the milk continues to be suckled by the young, milk production however ceasing very rapidly, the (3-Glu activity is not found to increase greatly during the first phase of involution. The behavior of the lysosomal enzymes during the second phase resembles that seen during normal involution.After administration of the ergot alkaloid 2-Br-a-ergokryptine methane sulfonate (CB 154), the process of involution is initiated only in individual mammary gland cells if the young are left with the mother. In these areas involution takes a normal course. Altogether, milk production changes only insignificantly.The cytostatic drug 5-fluorouracil does not induce direct involution. Milk production apparently is not arrested. However, the pups, when left with the mother, die after 5 to 6 days. Death would appear to be due to the cytotoxic action of 5-fluorouracil.     

Engineering Disease Resistant Cattle

Mastitis is a disease of the mammary gland caused by pathogens that find their way into the lumen of the gland through the teat canal. Mammary gland infections cost the US dairy industry approximately $2 billion dollars annually and have a similar impact in Europe. In the absence of effective treatments or breeding strategies to enhance mastitis resistance, we have created transgenic dairy cows that express lysostaphin in their mammary epithelium and secrete the antimicrobial peptide into milk. Staphylococcus aureus, a major mastitis pathogen, is exquisitely sensitive to lysostaphin. The transgenic cattle resist S. aureus mammary gland challenges, and their milk kills the bacteria, in a dose dependent manner. This first step in protecting cattle against mastitis will be followed by introduction of other genes to deal with potential resistance issues and other mastitis causing organisms. Care will be taken to avoid altering milk’s nutritional and manufacturing properties. Multi-cistronic constructs may be required to achieve our goals as will other strategies possibly involving RNAi and gene targeting technology. This work demonstrates the possibility of using transgenic technology to address disease problems in agriculturally important species. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Truncation of the beta-catenin binding domain of E-cadherin precedes epithelial apoptosis during prostate and mammary involution

A potential target of hormone action during prostate and mammary involution is the intercellular junction of adjacent secretory epithelium. This is supported by the long-standing observation that one of the first visible stages of prostate and mammary involution is the disruption of interepithelial adhesion prior to the onset of apoptosis. In a previous study addressing this aspect of involution, we acquired compelling evidence indicating that the disruption of E-cadherin-dependent adhesion initiates apoptotic programs during prostate and mammary involution. In cultured prostate and mammary epithelial cells, inhibition of E-cadherin-dependent aggregation resulted in cell death following apoptotic stimuli. Loss of cell-cell adhesion in the nonaggregated population appeared to result from the rapid truncation within the cytosolic domain of the mature, 120-kDa species of E-cadherin (E-cad(120)). Immunoprecipitations from cell culture and involuting mammary gland demonstrated that this truncation removed the beta-catenin binding domain from the cytoplasmic tail of E-cadherin, resulting in a non-beta-catenin binding, membrane-bound 97-kDa species (E-cad(97)) and a free cytoplasmic 35-kDa form (E-cad(35)) that is bound to beta-catenin. Examination of E-cadherin expression and cellular distribution during prostate and mammary involution revealed a dramatic reduction in junctional membrane staining that correlated with a similar reduction in E-cad(120) and accumulation of E-cad(97) and E-cad(35). The observation that E-cadherin was truncated during involution suggested that hormone depletion activated the same apoptotic pathway in vivo as observed in vitro. Based on these findings, we hypothesize that truncation of E-cadherin results in the loss of beta-catenin binding and cellular dissociation that may signal epithelial apoptosis during prostate and mammary involution. Thus, E-cadherin may be central to homeostatic regulation in these tissues by coordinating adhesion-dependent survival and dissociation-induced apoptosis.     

Mfge8 is critical for mammary gland remodeling during involution

Apoptosis is a critical process in normal mammary gland development and the rapid clearance of apoptotic cells prevents tissue injury associated with the release of intracellular antigens from dying cells. Milk fat globule-EGF-factor 8 (Mfge8) is a milk glycoprotein that is abundantly expressed in the mammary gland epithelium and has been shown to facilitate the clearance of apoptotic lymphocytes by splenic macrophages. We report that mice with disruption of Mfge8 had normal mammary gland development until involution. However, abnormal mammary gland remodeling was observed postlactation in Mfge8 mutant mice. During early involution, Mfge8 mutant mice had increased numbers of apoptotic cells within the mammary gland associated with a delay in alveolar collapse and fat cell repopulation. As involution progressed, Mfge8 mutants developed inflammation as assessed by CD45 and CD11b staining of mammary gland tissue sections. With additional pregnancies, Mfge8 mutant mice developed progressive dilatation of the mammary gland ductal network. These data demonstrate that Mfge8 regulates the clearance of apoptotic epithelial cells during mammary gland involution and that the absence of Mfge8 leads to inflammation and abnormal mammary gland remodeling.     

Epithelial cell-directed efferocytosis in the post-partum mammary gland is necessary for tissue homeostasis and future lactation

Background  

Mammary glands harbor a profound burden of apoptotic cells (ACs) during post-lactational involution, but little is known regarding mechanisms by which ACs are cleared from the mammary gland, or consequences if this process is interrupted. We investigated AC clearance, also termed efferocytosis, during post-lactational remodeling, using mice deficient for MerTK, Axl, and Tyro3, three related receptor tyrosine kinases (RTKs) regulating macrophage-mediated efferocytosis in monocytes. MerTK expression, apoptosis and the accumulation of apoptotic debris were examined in histological sections of MerTK-deficient, Axl/Tyro3-deficient, and wild-type mammary glands harvested at specific time points during lactation and synchronized involution. The ability of primary mammary epithelial cells (MECs) to engulf ACs was assessed in culture. Transplant of MerTK-deficient mammary epithelium into cleared WT mammary fat pads was used to assess the contribution of WT mammary macrophages to post-lactational efferocytosis.     

Evaluation of an anti-inflammatory factor derived from hyperimmunized cows

An anti-inflammatory factor isolated from milk of hyperimmunized cows was analyzed in vitro and in vivo. Macrophages collected from lacteal secretions of a unimmunized nonlactating cow showed increased ability to kill phagocytosed Staphylococcus aureus when incubated with the anti-inflammatory factor. Mice injected intraperitoneally with 10 mg/kg of anti-inflammatory factor demonstrated an increased LD50 to S. aureus when challenged intraperitoneally. Injected mice also demonstrated significantly (P less than 0.05) less mammary inflammation and involution and increased clearance of S. aureus when challenged intramammarily. Quantitative histologic analysis of mammary tissues from mice injected with anti-inflammatory factor demonstrated significantly (P less than 0.05) more lumen, less interalveolar connective tissue, and less leukocytic infiltration compared with control mice. Mammary glands of mice injected with anti-inflammatory factor and challenged with S. aureus also contained fewer colony-forming units than control mice. The product appeared to exert its effect on the nonspecific defense system via modulation of leukocyte function.     

The acute-phase protein serum amyloid A3 is expressed in the bovine mammary gland and plays a role in host defence

The serum amyloid A protein is one of the major reactants in the acute-phase response. Using representational difference analysis comparing RNA from normal and involuting quarters of a dairy cow mammary gland, we found an mRNA encoding the SAA3 protein (M-SAA3). The M-SAA3 mRNA was localized to restricted populations of bovine mammary epithelial cells (MECs). It was expressed at a moderate level in late pregnancy, at a low level through lactation, was induced early in milk stasis, and expressed at high levels in most MECs during mid to late involution and inflammation/mastitis. The mature M-SAA3 peptide was expressed in Escherichia coli, antibodies made, and shown to have antibacterial activity against E. coli, Streptococcus uberis and Pseudomonas aeruginosa. These results suggest that the mammary SAA3 may have a role in protection of the mammary gland during remodelling and infection and possibly in the neonate gastrointestinal tract.