Immunobiology of bovine mammary gland: apoptosis of somatic cells in milk during naturally occurring mastitis

Mastitis remains a major cause of economic losses in dairy herds. It is believed, that the enhancement of natural defense mechanisms in mammary gland may be helpful in the treatment of bovine mastitis. Our study was designed to assess the apoptosis of leukocytes isolated from bovine milk during subclinical staphylococcal mastitis. Milk samples were collected from cows naturally infected with one pathogen--Staphylococcus aureus and from animals with mastitis caused by several pathogens, including S. aureus. It has been determined that the rate of apoptosis was lower in mastitic milk, as compared with control samples, although varied significantly between groups. High percentage of apoptotic cells was detected in milk with high counts of pathogenic bacteria. In all groups the rate of apoptosis was dependent on the bacterial load, although various correlations were identified. Thus, it is postulated, that the rate of apoptosis of somatic cells in mastitic milk is related to the etiology of infection and is determined by the bacterial load.     

The complement in milk and defense of the bovine mammary gland against infections

The mammary gland of dairy cows, which is prone to infection by various bacteria, mobilizes local and systemic immune defenses to cope with pathogens. The complement system plays an important part in the innate immunity against microorganisms through its bactericidal, opsonic, and phlogistic functions. The amount of the complement in the milk of healthy glands of dairy cows is low. Moreover, the classical pathway of activation is not functional because of a shortage in C1q. By contrast, the alternative pathway is active, deposits C3b and C3bi on bacteria, and generates amounts of C5a which are highly variable among cows. A slight inhibition of the bactericidal/hemolytic activities, of the deposition of C1q on bacteria, and of the phlogistic activity of C5a makes milk a rather anti-inflammatory fluid. The inhibitory activity does not involve C3b/ C3bi deposition on bacteria, nor the generation of C5a by the alternative pathway. When inflammation develops, the blood-derived complement components overcome the inhibitions and complement-dependent bactericidal, opsonic and phlogistic activities may be high in milk. Further research is necessary to evaluate the contribution of C5a to the recruitment of leukocytes in the mammary gland, and to specify the links between the complement system and the response of resident cells (leukocytes and mammary epithelial cells) to infection stimulus. This will help to define the contribution of the complement system to resistance against mastitis, and could help to differentiate animals more or less resistant to this frequent and costly disease.     

Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk

Adiponectin is an adipocyte-derived hormone, which circulates in the form of homo-multimers. The individual oligomers have a distinct profile of activity, playing crucial roles in several biological processes, including metabolism and inflammation. Adiponectin exerts many of its effects by interacting with the receptors, AdipoR1 and AdipoR2. In the present study, mRNA expression of adiponectin, AdipoR1 and AdipoR2 was evaluated by quantitative PCR in different areas of the mammary gland in healthy lactating cows. The adiponectin isoforms in milk and blood were investigated by Western blotting and 2D-electrophoresis, and the presence of adiponectin protein was determined by immunohistochemistry.Low level expression of adiponectin mRNA was found in all areas of bovine mammary gland tissues examined. AdipoR1 and AdipoR2 mRNAs were also detected in mammary tissues and their expression was particularly prominent in the parenchyma and cistern. Western blotting revealed a heterogeneous electrophoretic pattern, indicating that different adiponectin isoforms exist in milk, compared with blood. In particular, milk shows a low molecular weight isoform of adiponectin, corresponding to the globular domain. Adiponectin in milk is characterised by a more complex 2D electrophoretic pattern, compared with blood, as illustrated by the presence of proteins of different molecular weights and isoelectric points. Adiponectin protein was detected by immunohistochemistry in epithelial cells lining the secretory alveoli, in secretum within the alveolar lumen and in small peripheral nerves. The study findings support a role for adiponectin in regulating metabolism and immunity of the bovine mammary gland and potentially the calf intestine, following ingestion of milk.     

Morphologic changes in the bovine mammary gland during involution and lactogenesis

Morphologic changes developing during bovine mammary involution were examined. Quarter biopsy specimens were obtained weekly from 5 cows beginning the day milking was discontinued through parturition. Light and electron microscopic examination of mammary tissue indicated a gradual reduction in synthetic and secretory activity of alveolar epithelium as involution progressed. Light microscopic morphologic analysis revealed increases in stroma and nonactive secretory epithelium, with concomitant decreases in epithelium, lumen, and fully active secretory epithelium during the first 2 weeks of involution. Electron microscopic analysis of alveolar epithelium revealed decreased number of organelles associated with milk synthesis and secretion during this time. These changes reversed gradually beginning 2 weeks before parturition, and by the time of calving, cell structure was typical of lactating glands. Tissue from infected quarters had less synthetic and secretory ability as indicated by significantly higher percentages of stroma and nonactive cells, but lower percentages of lumen and moderately active cells, compared with uninfected quarters. Infected quarters also had more leukocytes infiltrating the epithelium, lumen, and stroma, compared with uninfected quarters. Microscopic examination of macrophages and neutrophils suggested these cells removed milk components and cellular debris during involution. Large numbers of plasma cells, with distended cisternae of rough endoplasmic reticulum, suggested local antibody production during the periparturient period.     

Cytokines in mammary lymph and milk during endotoxin-induced bovine mastitis

Cytokine kinetics were examined in milk and in afferent and efferent lymph of the supramammary lymph node after intramammary infusion of endotoxin from Escherichia coli. Cows were sampled 0, 2 and 4h after infusion (p.i.). Neutrophils appeared in afferent lymph 2h p.i., and in efferent lymph and milk 4h p.i. The milk contained high concentrations of interleukin-8 (IL-8) at 2 and 4h p.i. IL-8 was also found in lymph, but at lower concentrations. The tumor necrosis factor-alpha (TNF-alpha) concentration tended to increase in afferent lymph at 2h p.i., and increased in milk at 4h p.i. The level of IL-1beta increased at 4h p.i. in milk, but was not detected in lymph. Interferon-gamma was not detected in any sample, at any time. The results indicate a primary role for IL-8 in the recruitment of neutrophils into the gland, and suggest that IL-1beta and TNF-alpha are not necessary for IL-8 production and release in response to endotoxin.     

Neutrophil apoptosis during the resolution of bovine mammary gland injury

The role of neutrophil apoptosis in the resolution of bovine mammary gland injury induced by intramammary administration of physiological buffered saline (PBS) or lipopolysaccharide (LPS) was investigated. Twenty mammary glands of five non-pregnant heifers were used in the two studies and each animal received both stimuli. Samples of cell populations were collected by mammary gland lavages before and 24, 48, 72 and 96 hours after treatment and examined by light microscopy and staining for myeloperoxidase (MPO). A marked influx of neutrophils into the mammary gland was observed 24 hours after stimulation. At the same time, apoptotic neutrophils and MPO-positive macrophages (MAC) were identified in the samples. The numbers increased to reach maximum values at 48 hours after stimulation with PBS and at 72 hours after stimulation with LPS. The observed differences in the length of the resolution period indicate that neutrophil viability can be modulated by delaying the apoptotic process. Apoptosis of neutrophils and their subsequent phagocytosis by MAC can be regarded as a significant mechanism in the removal of neutrophils from the acutely injured mammary glands and, hence, in the resolution of bovine mastitis.     

Defences of the bovine mammary gland against infection and prospects for their enhancement

The equine alternative complement pathway has been partially characterized and compared to the equine classical activation pathway. A dose-dependent lysis of RbRBC was observed with peak lytic values noted within 10 minutes at 37°C when rabbit red blood cells (RbRBC) were used as an alternative pathway activator. Sheep red blood cells (SRBC) sensitized with rabbit hemolysin or partially purified equine IgM antibodies were equally sensitive to lysis. Dilution of the commercial hemolysin by $\text{1}\text{5}$ reduced lysis from 90% to 38% in the presence of constant cell numbers. Hemolysis of SRBC peaked at 10 minutes and the majority of lysis occurred within 10 minutes. Dilution of equine sera by as little as $\text{1}\text{5}$ decreased hemolytic activity for SRBC to 21.5% from greater than 90% with undiluted sera. The alternative pathway protein, equine factor B, was tested using RbRBC and monitored by its differential susceptibility to heat treatment at 50°C. This treatment led to almost complete inactivation after a 15-minute incubation. An apparent heat-dependent decay of certain classical pathway components was also observed after 50°C treatment. This sensitivity was indicated by a reduction in the lytic activity for sensitized SRBC. Treatment for 15 minutes at 56°C with either RbRBC or SRBC was sufficient to abolish hemolytic activity in all equine sera tested. Chelation of cations with 0.04 M EDTA blocked expression of alternative and classical pathway activation; however, chelation of Ca⁺⁺ ions with 10 mM EGTA containing 1 mM Mg⁺⁺ ions permitted lysis of the RbRBC but not the SRBC. A dose-related Mg⁺⁺-ion dependence for RbRBC hemolytic activity was observed as the concentration of Mg⁺⁺ was increased to 1.0 mM. In addition, our results obtained with pre-colostral foal serum strongly suggest that natural antibody to RbRBC was of little importance in the lysis observed with these cells. These results also show that the equine alternative pathway activation may require Ca⁺⁺ ions. If Ca⁺⁺ ions are required, the equine alternative pathway is quite different from any other mammalian complement system so far described. Our results suggest that the alternative pathway of activation is of major importance in the equine complement system. Confirmation of this hypothesis requires both purification of the components involved as well as further characterization.     

Cytokine activity in bovine mammary gland secretions during the periparturient period.

The presence of cytokine activity in periparturient bovine mammary secretions was evaluated. Mammary secretions were modified for use in biological assays for interleukin-2 (IL-2) like and antiviral activity. The level of IL-2 like activity in mammary gland secretions was lower during the last week of gestation when compared to levels detected approximately two weeks prepartum. Antiviral titers gradually increased as parturition approached. Results from Western blots indicated that the antiviral activity observed in prepartum secretions may be due to tumor necrosis factor (TNF). Interferons (IFN) were not detected in the colostrum samples.     

与脐带间充质干细胞共培养对奶牛乳腺上皮细胞乳脂合成及关键基因表达的影响

探究与脐带间充质干细胞共培养对奶牛乳腺上皮细胞(BMECs)乳脂合成及关键基因表达的影响。将脐带间充质干细胞和乳腺上皮细胞利用Transwell小室双层共培养,BMECs单纯培养为对照组,IGF-ⅠR抑制剂AG1024处理细胞,检测上清IGF-Ⅰ、甘油三酯(TAG)含量变化,再用磷脂酰肌醇-3-羟激酶(PI3K)信号阻断剂LY294002孵育细胞,RT-qPCR检测乙酰辅酶A羧化酶(ACACA)、脂肪酸合成酶(FASN)和固醇调节元件结合蛋白(sterol regulatory element.binding proteins,SREBP1)基因的相对表达丰度。结果显示,共培养后BMECs的IGF-I含量极显著升高(P0.01),TAG含量显著升高(P0.05);加入AG1024后,IGF-I明显受到抑制(P0.01),显著降低了各组TAG含量及各基因的表达丰度(P0.05);LY294002抑制了PI3K(P0.01)、AKT、mTOR(P0.05)mRNA的表达,显著降低了TAG含量及ACACA、FASN、SREBP1mRNA的表达(P0.05);共同处理后极显著降低了TAG合成量及各基因相对表达丰度(P0.01)。结果表明,脐带间充质干细胞能够通过IGF-Ⅰ介导PI3K/Akt/mTOR信号通路上调BMECs乳脂合成关键基因的表达丰度,促进TAG的合成。     

Biopsy of the bovine mammary gland.

A technique is described for biopsy of the bovine udder, employing sedation and local anaesthesia. Tissue samples of approximately 5 g were obtained by electrocautery from two quarters of the udder of a cow laterally recumbent. Care was taken to ensure complete haemostasis which was achieved by electrocoagulation and ligation. Postoperative recovery was rapid, and loss of yield was no greater in biopsied glands than in control glands of the same cow. Yield from all quarters returned to preoperative levels within 48 h.     

Induced staphylococcal infections in the bovine mammary gland

In a study to develop and define a practical model of bovine mastitis caused by Staphylococcus aureus, induced infections were attempted in 203 bovine mammary glands of 41 cows, using 12 strains of S aureus. Approximately 100 colony-forming units of S aureus in saline solution were injected after milking, and milk samples were collected daily from test glands for 14 days to monitor the progress of infections and inflammatory responses. Relationships were examined for cow-related factors and for various characteristics of the strains of S aureus used to the development of a persistent intramammary infection. A dairy cow that was useful in this model was defined as follows: (1) the 2nd to 7th month in the 1st to 5th lactation; (2) producing milk from all mammary glands that contained less than 6 x 10(5) somatic cells/ml; and (3) having mammary glands that were free of any primary mastitis pathogen, as well as micrococci and Corynebacterium bovis. From the present study, it was not possible to define clearly a strain of S aureus which would be useful in the model, but 5 strains of S aureus were identified as being capable of producing persistent subacute infections with a high degree of repeatability.     

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

Although the functions of adiponectin, a differentiated adipocyte‐derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry‐off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.     

Apoptosis of polymorphonuclear leukocytes of the juvenile bovine mammary gland during induced influx

The dynamics of apoptosis of polymorphonuclear leukocytes (PMN) during induced influx of PMN into the cavity system of the juvenile bovine mammary gland in order to investigate the role of apoptosis of PMN in the resolution of mastitis was studied. The instillation of a synthetic analogue of muramyl dipeptide into teat sinus of the sixteen mammary glands was followed by a massive influx of PMN culminating after 24 h and resolving after 96 h. Every 24 h following the influx, apoptotic PMN were microscopically detected, based on morphological characteristics. Twenty four hours after the stimulation, apoptotic PMN were already observed, and peak counts of apoptotic PMN were reached 48 h after the stimulation. The lowest differential count of apoptotic PMN, corresponding to the pre-stimulation value, was found 96 h after the stimulation. The presence of macrophages (MAC) containing phagocytized apoptotic PMN was observed by histochemical staining for myeloperoxidase (MPO) and electron microscopy. The percentage of MPO-positive macrophages increased during the resolution phase to reach peak values 48 h after the stimulation. Apoptosis of PMN and phagocytosis by macrophages may represent a removal mechanism that is important in the resolution of the induced influx of PMN in the cavity system of juvenile bovine mammary gland.     

Expression of bone morphogenetic protein-6 and bone morphogenetic protein receptors in myoepithelial cells of canine mammary gland tumors

To study the ectopic chondrogenesis in canine mammary mixed tumors, the expression of bone morphogenetic protein-6 (BMP-6) and specific BMP receptors (BMPRs), BMPR-IA, BMPR-IB, and BMPR-II, was examined using immunohistochemical and immunoblot analysis in 39 canine mammary gland tumors. Immunohistochemically, BMP-6 and all three types of BMPRs were coexpressed in the myoepithelial cells and chondrocytes in six of eight benign mixed tumors. In complex adenomas, myoepithelial cells showed an expression pattern of BMP-6, BMPR-IA, and BMPR-II similar to those in benign mixed tumors, whereas immunoreactivity for BMPR-IB was very mild. The myoepithelial cells proliferating within the basement membrane showed more intense immunoreactivity for BMP-6 and all BMPRs as compared with those proliferating in the interstitial areas. Western blotting analysis revealed immunopositive bands at 40-45 kDa for BMP-6 in the samples from simple and complex adenomas and benign mixed tumors. The BMPR-IB-specific bands at 45 kDa were most detected in benign mixed tumors. Because among BMPRs, BMPR-IB is thought to be the major receptor for BMP-6 for primary chondrogenesis, these findings suggest that the expression of BMP and its receptors on the myoepithelial cells might play a role in the ectopic cartilage formation in canine mammary gland tumors, especially in benign mixed tumors.     

Effects of Staphylococcus aureus mastitis on bovine mammary gland plasma cell populations and immunoglobin concentrations in milk

Bovine mammary tissue and milk samples were examined to determine effects of chronic Staphylococcus aureus mastitis on the humoral immune response. Parenchymal and teat end tissues from lactating bovine mammary glands were stained immunohistochemically to determine distribution of immunoglobulin (Ig) G1-, IgG2-, IgA-, and IgM-producing plasma cells. Numbers of all Ig-producing plasma cells tended to be higher in tissues from S. aureus infected quarters compared with controls, but most differences were not statistically different. Numbers of IgG1-producing plasma cells at the Furstenberg's rosette area of infected quarters were significantly (P less than 0.05) higher than uninfected quarters. There were no significant differences in concentrations of Ig isotypes in milk from S. aureus infected and uninfected quarters. Data suggest that the antigenic effect of chronic S. aureus infection on the humoral immune response of the bovine mammary gland is minimal. Persistency of S. aureus infection may result, in part, from suboptimal stimulation or immunosuppression of the mammary immune system.