Apoptosis of resident and inflammatory macrophages before and during the inflammatory response of the virgin bovine mammary gland

Background

Macrophages may play a prominent role in defense of the bovine mammary gland, and their functionality is necessary for successful eradication of bacterial pathogens. In contrast to necrosis, however, apoptosis has not yet been studied in macrophages from bovine mammary glands. Therefore, the aim of this study was to confirm the occurrence of apoptosis in macrophages from resting heifer mammary glands and during the inflammatory response.

Methods

Inflammatory response was induced by phosphate buffered saline (PBS) and by lipopolysaccharide (LPS). Resident macrophages (_RESMAC) were obtained before and inflammatory macrophages (_INFMAC) 24, 48, 72 and 168 hours after inducing inflammatory response in mammary glands of unbred heifers. Cell samples were analyzed for differential counts, apoptosis and necrosis using flow cytometry.

Results

Populations of _RESMAC and _INFMAC contained monocyte-like cells and vacuolized cells. Apoptosis was detected differentially in both morphologically different types of _RESMAC and _INFMAC and also during initiation and resolution of the inflammatory response. In the _RESMAC population, approximately one-tenth of monocyte-like cells and one-third of vacuolized cells were apoptotic. In the _INFMAC population obtained 24 h after PBS treatment, approximately one-tenth of monocyte-like cells and almost one-quarter of vacuolized cells were apoptotic. At the same time following LPS, however, we observed a significantly lower percentage of apoptotic cells in the population of monocyte-like _INFMAC and vacuolized _INFMAC. Moreover, a higher percentage of apoptotic cells in _INFMAC was detected during all time points after PBS in contrast to LPS. Comparing _RESMAC and _INFMAC, we observed that vacuolized cells from populations of _RESMAC and _INFMAC underwent apoptosis more intensively than did monocyte-like cells.

Conclusions

We conclude that apoptosis of virgin mammary gland macrophages is involved in regulating their lifespan, and it is involved in the resolution process of the inflammatory response.     

The bovine neutrophil: Structure and function in blood and milk

Migration of polymorphonuclear neutrophil leukocytes (PMN) into the mammary gland provide the first line of defense against invading mastitis pathogens. Bacteria release potent toxins that activate white blood cells and epithelial cells in the mammary gland to secrete cytokines that recruit PMN that function as phagocytes at the site of infection. While freshly migrated PMN are active phagocytes, continued exposure of PMN to inhibitory factors in milk such as fat globules and casein, leads to altered PMN morphology and reduced phagocytosis. In the course of phagocytosing and destroying invading pathogens, PMN release chemicals that not only kill the pathogens but that also cause injury to the delicate lining of the mammary gland. This will result in permanent scarring and reduced numbers of milk secretory cells. The life span of PMN is limited by the onset of apoptosis. To minimize damage to mammary tissue, PMN undergo a specialized process of programmed cell death known as apoptosis. Macrophages quickly engulf and phagocytose apoptotic PMN, thereby minimizing the release of PMN granular contents that are damaging to tissue. The PMN possess an array of cell surface receptors that allow them to adhere and migrate through endothelium and to recognize and phagocytose bacteria. One receptor found on phagocytes that is receiving considerable attention in the control of infections by Gram-negative bacteria is CD14. Binding of lipopolysaccharide (LPS) to membrane bound CD14 causes release of tumor necrosis factor-alpha and sepsis. Binding of LPS to soluble CD14 shed from CD14-bearing cells results in neutralization of LPS and rapid recruitment of PMN to the site of infection. Recent advances in the fields of genomics and proteomics should greatly enhance our understanding of the PMN role in controlling intramammary infections in ruminants. Further, manipulation of PMN, through either recombinant proteins such as soluble CD14 that enhance PMN response or agents that mediate PMN apoptosis, may serve as novel therapeutics for the treatment of mastitis.     

Effect of apoptosis on phagocytosis, respiratory burst and CD18 adhesion receptor expression of bovine neutrophils

Polymorphonuclear neutrophil leukocytes (PMN) play an important role in intramammary defense against infections by Escherichia coli. During mastitis, PMN are confronted with various inflammatory mediators that can modulate their function. In severely diseased cows, increased concentrations of lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha (TNF-alpha) are detected in plasma. Binding of LPS to membrane bound CD14 molecules on monocytes cause release of inflammatory mediators such as TNF-alpha. Because apoptosis of PMN promotes resolution of inflammation and because the LPS and TNF-alpha response in milk and blood is related to the severity of E. coli mastitis, the effect on apoptosis of bovine PMN of increased concentrations LPS and TNF-alpha was studied together with the functionality of apoptotic PMN.Bovine PMN apoptosis, as determined with annexin-V, was induced with high concentrations of either LPS (1000 and 10,000ng/mL) or TNF-alpha (10,000ng/mL) in whole blood following a 6h incubation at 37 degrees C. The apoptosis inducing effect of LPS on PMN was not inhibited following coculture with either anti-bovine TNF-alpha or anti-ovine CD14 monoclonal antibodies. When compared to controls, apoptotic PMN had a similar level of CD18 expression but lacked phagocytic and respiratory burst activity. This is the first study reporting the effects of apoptosis on bovine PMN function. These functional impairments in apoptotic PMN could be important in contributing to the establishment of intramammary infection. Well functioning PMN could finally determine the severity of mastitis following an invasion of bacteria in the mammary gland.     

Apoptosis and necrosis of blood and milk polymorphonuclear leukocytes in early and midlactating healthy cows.

Increased milk somatic cell counts (SCC) are used as an indicator for bovine mastitis. During mastitis, polymorphonuclear leukocytes (PMN) become the predominant cell type. Shortly after parturition, the severity of mastitis is increased and several PMN functions are downregulated. Apoptotic and necrotic processes of PMN could influence SCC and PMN functions. In this study, the percentages of apoptotic and necrotic PMN in blood and milk from early and midlactating healthy cows were compared. Apoptosis and necrosis of PMN were quantified using a dual-color flow cytometric procedure with fluorescein labeled annexin-V (green) and propidium iodide (red). Using this technique three different subpopulations of bovine PMN could be detected: apoptotic cells (high intensive green fluorescence), necrotic cells (high intensive green and high intensive red fluorescence) and viable cells (low intensive green and low intensive red fluorescence). Following a 4 h incubation of blood from both groups of cows at 37 degrees C to induce apoptosis, the mean percentage of apoptotic blood PMN was significantly higher (P < 0.01) in early lactating cows (15.1%, n = 9) compared with midlactating cows (5.3%, n = 10). The mean percentage of necrotic PMN remained lower than 5% in all cows. In contrast to blood, no significant difference was found between the percentage of apoptotic PMN in milk from early (41.2%, n = 7) and midlactating cows (34.0%, n = 8). The percentage of necrotic PMN in milk from early lactating cows (25.9%, n = 7) was significantly higher than that in midlactating cows (14.2%, n = 8) (P < 0.05). Higher percentages of apoptotic as well as necrotic PMN were consistently found in milk compared to blood in all cows. From these results, it can be concluded that spontaneously induced apoptosis was higher in blood PMN from early lactating cows than in blood PMN from midlactating cows. The higher percentage of necrotic milk PMN in early lactating cows than in midlactating cows could be explained by the induction of secondary necrosis.     

CD44 is highly expressed on milk neutrophils in bovine mastitis and plays a role in their adhesion to matrix and mammary epithelium

Mastitis, inflammation of the mammary gland, is a common and economically important disease in dairy animals. Mammary pathogenic organisms, such as Escherichia coli, invade the teat canal,milk ducts, and mammary alveolar space, replicate in mammary secretions, and elicit a local inflammatory response characterized by massive recruitment of blood polymorphonuclear neutrophil leukocytes (PMN) into the alveoli and milk ducts. CD44 is a trans-membrane glycoprotein previously shown to play a role in mediation and control of blood PMN recruitment in response to inflammatory signals. Here we show, for the first time, increased expression of CD44 on recruited milk PMN in bovine mastitis and the expression of a CD44 variant, CD44v10, on these PMN. Furthermore, we demonstrate that CD44 mediates specific adhesion of bovine blood PMN to hyaluronic acid and mammary epithelial cells. Our results suggest that in mastitis CD44 plays a role in recruiting blood PMN into the mammary glands, the exact nature of this role needs to be elucidated.     

Blood and milk cellular immune responses of mastitic non-periparturient cows inoculated with Staphylococcus aureus

Lymphocyte function and phenotype of peripheral blood and mammary gland cells were evaluated in non-periparturient cows before and at 1, 4 to 8 and 9 to 14 d after inoculation with Staphylococcus aureus, as expressed by percentage of CD3+, CD2+, and CD45R+ cells, antigen density of these markers per lymphocyte, and mitogen-induced blastogenesis. Milk bacterial counts and somatic cell counts (SCC) were also assessed. Mitogen-induced blastogenic responses were strong in blood and weak in mammary gland cells in all observations and positively correlated with the percent of CD45R+ cells. Significantly greater percentages of milk CD3+ lymphocytes and increased CD3, CD2, and CD45R antigen density per cell were observed after challenge. The blood CD3 and CD2 antigen density per lymphocyte and the milk CD2+ lymphocyte percent were negatively correlated with SCC (P ≤ 0.01). No mastitis (SCC ≤ 500 000 cells/mL) was observed in cows showing blood lymphocyte CD2 and CD3 antigen density indices ≥ 2.5 and 6, respectively. Forty-one percent of SCC values were predicted by the combined blood CD2 and milk CD3 antigen density (P ≤ 0.01). These findings support the hypotheses that mitogen-induced lymphocyte blastogenesis is not a valid test to assess mammary gland immunocompetence and that CD2 expression may facilitate immune responses by decreasing the number of T cell receptors required to achieve full activation.     

Phagocytosis and Related Phenomena in Polymorphonuclear Leukocytes from Cow's Milk

There were fewer efficient phagocytes among leukocytes collected from artificially irritated mammary glands than among the leukocytes from blood of the same animals. The milk polymorphonuclear (PMN) leukocytes adhered poorly to a column of siliconised glass beads when compared with the blood cells. However, investigations of the O₂ uptake and CO₂ production of the milk PMN leukocytes revealed that these cells appeared to utilize metabolic pathways similar to those used by human and guinea pig PMN leukocytes during phagocytosis. These pathways are associated with degranulation and the production of H₂O₂ following particle ingestion. It is therefore suggested that the milk PMN leukocytes appear not to have lost the ability to produce this bactericidal substance.     

Immune cell differentiation in mammary gland tissues and milk of cows chronically infected with Staphylococcus aureus

This study identifies and compares the distribution of mononuclear cells in the mammary gland tissues and milk of healthy and chronically infected with Staphylococcus aureus cows. Somatic cell counts (SCCs) during the 3 months before the study were > 1 x 10(6) cell/ml in the infected quarters and < 1 x 10(5) cell/ml in the infection-free quarters. Immediately after slaughter, samples from the tissues above the gland cistern and supra-mammary lymph node were collected. No histological differences were found between the supra-mammary lymph nodes of the healthy and infected udders, and both appeared normal. In the milk of the healthy infection-free mammary glands, SCC was < 50,000 cells/ml) while epithelial cells were the predominant type. The percentage of CD18+ was low than 45%, of which over three-quarters were polymorphonuclear (PMN), and less than one- quarter were mononuclear cells. The later comprised CD4+ or CD8+ T-lymphocytes, macrophages (Mo) but not B-cells. In the tissues, there were few CD18+ leukocytes, and most of the cells were T-lymphocytes. The number of B-lymphocytes bearing CD21+ was similar to that of CD8+ and were localized in the connective tissue as clusters of 2-5 cells, mainly in areas with no alveoli, or as single cell having a dendritic like form. The number of Mos was negligible. In the milk of the infected glands, SCC exceeded 700,000 cells/ml, of which > 95% were CD18+ positive. The distribution of the leukocytes had two patterns: one presented (> 80%) of PMN cells and a small number of mononuclear cells; the second had less than 50% PMN and many mononuclear cells. The CD8+ cells in these infected sections were observed throughout the mammary epithelial cells (MEc) around the alveoli and in the alveolar lumen (AL). The numbers and the location of CD21+ B-lymphocytes were similar to those in the infection-free mammary glands. The number of CD5+ positive cells was lower than T and B- cells combined and were located throughout the mammary epithelial cells, around the alveoli and within the connective tissue. Mo numbers were high in most of those infected quarters, and were localized around the connective tissue and within the AL.     

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.     

Effectiveness of small interfering RNA (siRNA) against the Mcl-1 gene in a canine mammary gland tumor cell line

The effect of down-regulation of Mcl-1 expression by small interfering RNA (siRNA) against the canine Mcl-1 gene on apoptosis was investigated by transfecting CF33 (canine mammary gland tumor cell line) with siRNA using cationic liposomes. The siRNA against canine Mcl-1 increased the rate of apoptotic cells and decreased the numbers of viable cells. Further, sequence-specific down-regulation of Mcl-1 expression was measured by real time-PCR and Western blot analysis. The siRNA directed against the Mcl-1 gene reduced both the mRNA and protein expression in the CF33. Our study suggests the importance of Mcl-1 in canine mammary tumors for inducing apoptosis and reinforces using Mcl-1 as a putative therapeutic target in canine mammary gland tumor.     

Analysis of reporter gene expression in ovine dermis and afferent lymph dendritic cells in vitro and in vivo

A flow cytometric technique was used to detect apoptosis and necrosis of bovine polymorphonuclear neutrophil leukocytes (PMN) using fluorescein isothiocyanate labeled annexin-V and propidium iodide (PI). Isolation of PMN from the blood following lysis by water or NH4Cl resulted in false positive results for apoptosis. Therefore, a method was developed to identify living, apoptotic and necrotic PMN simultaneously in a single 100 microl blood sample. To establish a positive control for PMN apoptosis, the effect of cycloheximide, actinomycin D, diamide, buthionine sulfoximine and sodium arsenite, that have been described to induce apoptosis by various mechanisms was tested. Only actinomycin D induced a significant increase in the percentage of apoptotic PMN after 2 h. Incubation of blood for 6 h with cycloheximide, actinomycin D and buthionine sulfoximine resulted in a significant increase of apoptotic PMN compared to control values. Sodium arsenite, mainly caused necrosis after 6 h of incubation.     

Activation of phagocytes during initiation and resolution of mammary gland injury induced by lipopolysaccharide in heifers

The object of the study was the comparative assessment of phagocyte activation during initiation and resolution of mammary gland injury induced by lipopolysaccharide (LPS) or buffered salt solution (PBS) on the basis of the CD14 receptor positivity. The experiments were carried out in 15 clinically normal Holstein x Bohemian Red Pied crossbred heifers, aged 14 to 18 months. Noninflammatory and inflammatory mammary gland injury were induced by intramammary administration of PBS (10 mL) and LPS (10 mL, 1 microg/mL), respectively. Samples of the cell populations were obtained by mammary lavages at 24 h intervals. Flow cytometry was used to determine the CD14+ neutrophils, monocytes, and macrophages. The percentage of CD14+ neutrophils was only 1.2% and 1.3% 24 h after the treatment with PBS and LPS, respectively. The resolution was accompanied by an increase in proportion of CD14+ neutrophils. The proportion of CD14+ neutrophils returned to initial values in the PBS-treated, but not in the LPS-treated mammary glands till 96 h. Percentage of CD14+ monocytes increased after 24 h and the effect was more pronounced in the LPS-treated than in the PBS treated mammary glands (P < 0.05). The percentage of CD14+ macrophages decreased highly significantly at 24 h in the LPS-treated, but not in the PBS-treated mammary glands (P < 0.01). The resolution of mammary gland injury (48 to 96 h) was characterised by an increase in CD14+ macrophages proportion, which was greater in the LPS-treated than PBS-treated mammary glands (P < 0.01). The activation of macrophages during resolution of mammary gland injury can be interpreted as an important mechanism of restitution.     

The role of CD14 during resolution of experimentally induced Staphylococcus aureus and Streptococcus uberis mastitis

This study was undertaken to investigate the time course of surface expression of CD14 on neutrophils and macrophages and to determine their association with resolution of inflammatory responses during Staphylococcus aureus and Streptococcus uberis experimental mastitis. Infections of the mammary gland induce a local immune response characterized by an increase in the total counts of CD14+ neutrophils and CD14+ macrophages particularly. On the other hand, resolution is accompanied by an increase in relative counts of CD14+ neutrophils, CD14+ vacuolized macrophages and apoptotic neutrophils. Following the immune reaction of mammary gland against Gram-negative/positive bacteria is very similar. Between the apoptotic and CD14+ neutrophils a high correlation was measured during the whole experimental period (S. aureus: r=0.64; S. uberis: r=0.61; P<0.05). Using anti-CD14 monoclonal antibodies in vitro suggested the involving of the CD14 surface receptor in recognition of apoptotic neutrophils by macrophages.     

Variations among unbred heifers in the activities of polymorphonuclear leucocytes from the mammary gland and blood

The phenotypic characteristics are described for the activity of polymorphonuclear leucocytes NMN) obtained by either lavage of the cavity system of juvenile mammary glands stimulated with a synthetic muramyl dipeptide analogue or isolation from the peripheral blood. Attention was paid to the variability of characteristics and its sources, and to correlations among them. The following characteristics were investigated in 27 clinically healthy, unbred Bohemian Red Pied x Holstein heifers: migration activity in situ, number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and unstimulated and zymosan-stimulated luminol-dependent chemiluminescence. Considerable individual variation was found in the characteristics. Significant differences between blood PMN and PMN from lavages after influx induction were found for bactericidal activity (P < 0.05) and chemiluminescence (P < 0.01). A significant correlation between blood PMN and mammary gland PMN was found only for the number of phagocytosing cells (r = 0.329; P < 0.01). Highly significant positive correlations (P < 0.01) were demonstrated between the number of phagocytosing PMN [a], phagocytotic index [b], and bactericidal activity [c] in both blood PMN (r(ab) = 0.602; r(ac) = 0.565; r(bc) = 0.529) and mammary gland PMN (r(ab) = 0.730, r(ac) = 0.618, r(bc) = 0.589). No significant correlation was demonstrated for non-stimulated (NS), zymosan-stimulated (ZS), or opsonized zymosan-stimulated (OZS) chemiluminescence with any of the other characteristics of phagocytotic activity, in either blood PMN or mammary gland PMN (P > 0.05). The animal was a highly significant source of variability for all the phagocytotic activity characteristics (P < 0.01). Udder quarter was a non-significant source of variability for all the characteristics of phagocytotic activity except for NS chemiluminescence (P < 0.05) and ZS or OZS chemiluminescence (P < 0.01). However, udder quarter was a non-significant source of variability of chemiluminescence indices ZS/NS and OZS/NS (P > 0.05). It has been demonstrated that in situ migration activity, the number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and chemiluminescence indices of PMN collected from juvenile mammary glands of unbred heifers after influx induction can be regarded as candidate early markers of resistance to mammary infections.     

Studies on the Modulation of Leucocyte Subpopulations and Immunoglobulins following Intramammary Infusion of β1,3‐glucan into the Bovine Udder during the Dry Period

Inflammatory and immunological reactions after intramammary infusion of β1,3‐glucan were studied during the steady dry period and involution phase of the bovine udder. The effects of a single intramammary infusion of two different doses (100 and 200 mg) of β1,3‐glucan were evaluated during the steady dry period. In a second study, the effects of β1,3‐glucan at drying off were studied by using two treatment regimens; a single infusion at drying off, compared with two infusions of the compound, at drying off and again 2 weeks later. Total and differential leucocyte counts were measured in both blood and udder secretions. Additionally, the expression of receptors for CD14 and MHC class II on leucocytes, and the expression of receptors for CD4, CD8, WC1, IL2R and B‐cells on lymphocytes was measured in mammary secretions by flow cytometric analyses. The concentrations of immunoglobulins in udder secretions were measured by radial immunodiffusion. The results showed that a single intramammary infusion of β1,3‐glucan during the steady dry period causes transient enhancement of some aspects of the inflammatory and immune responses. The increases in somatic cell counts, numbers of monocytes/macrophages, and in proportions of CD14 + and MHC class II + leucocytes in udder secretions were dose‐dependent. Infusion of β1,3‐glucan also slightly increased the proportion of CD4 + lymphocytes and the concentrations of IgG₁ and IgG₂ in dry secretions. Infusion of β1,3‐glucan at drying off seemed to accelerate the involution process through an increase in somatic cells, particularly in the numbers of macrophages, in mammary secretions. The numbers of lymphocytes and polymorphonuclear leucocytes, the proportions of IL2R + lymphocytes, the proportions of CD14 + or MHC class II + leucocytes and the concentrations of IgG₁ and IgG₂ also increased in comparison with untreated controls. Moreover, a second infusion of β1,3‐glucan tended to prolong this response, indicating that this might be an effective means of enhancing the mammary defence against udder infections closer to calving. In conclusion, the results indicate that intramammary infusion of β1,3‐glucan could be used to enhance the defence mechanisms of the bovine udder against infections, especially during early involution.     

Effect of phytohaemagglutinin-P on apoptosis and necrosis in Trichinella spiralis infected mice

Flow cytometry analyses were used to evaluate the contribution of apoptotic and necrotic lymphocytes in the selected organs of Trichinella spiralis infected mice treated with phytohaemagglutinin-P (PHA-P). The Tunnel method was used to examine apoptosis in a cryostat section from the jejunum and masseter muscle. CFW mice (Groups I and II) were infected with 200 larvae of T. spiralis. PHA-P was administered intravenously at a dose of 10 mg/kg 24 h prior to infection in Group II mice only. Group III mice were treated with PHA-P without T. spiralis infection, and Group IV mice were untreated controls. The lymphocytes obtained from the spleen, mesenteric lymph nodes (MLN) and muscular inflammatory infiltration on 7, 14, 21, 28, 35, 42 and 60 days post infection (DPI) were incubated with the Annexin-V-Fluos Staining Kit (Roche). The cryostat preparation made from the jejunum and masseter muscle was evaluated using a fluorescence microscope. PHA-P administration stimulated apoptosis in the jejunal mucosa and in the muscular inflammatory infiltration. In Group I mice, infected with T. spiralis only, the highest percentage of apoptotic cells was found on 7 DPI in the spleen and in MLN, and on 14 DPI among the cells of the muscular inflammatory infiltration. The peak of the necrotic lymphocytes was found on 7 DPI in the spleen, on 28 DPI in MLN, and on 21 DPI in the cells of muscular inflammatory infiltration. In Group II mice, infected with T. spiralis and treated with PHA-P, the peak in apoptotic cells occurred on 7 DPI in the spleen and in the muscular inflammatory infiltration. The highest level of necrotic lymphocytes was observed only on 7 DPI in the muscular inflammatory infiltration. Percentage of necrotic lymphocytes in the spleen was the same and in MLN it was lower than in Group I (T. spiralis only). Moreover, the number of muscle larvae in mice treated with PHA-P (Group II) was lower than in Group I (T. spiralis only).     

Adhesion molecules and lymphocyte subsets in milk and blood of periparturient Holstein cows.

Migration of leukocytes into the mammary gland is an essential element of resistance to infection which is likely influenced by expression of adhesion molecules. The contribution of subsets to mammary gland resistance remains unclear. Mononuclear cells from milk and blood of dairy cows were examined for variation in CD4+, CD8+, and WC1+ (Workshop Cluster 1; marker for gammadelta T cells) lymphocyte phenotypes and expression of LFA-1 and L-selectin at several time points during the periparturient period and at Week 16 of lactation. Proportions of CD4+ T cells were higher (p < or = 10.05) in blood than milk at all times between Week 0 and Week 16 relative to calving; the inverse was true of CD8+ cells. Expression of L-selectin was lower (p < or = 0.05) on CD4+ cells and higher on CD8+ cells from milk. The WC1+ subset was more frequent in blood than in milk except at calving when the opposite was true. After calving, proportions of L-selectin+ WC1+ cells decreased steadily to Week 16. Expression of LFA-1 was examined on mononuclear cell populations and found to be lower on milk cells and did not vary over time. We conclude that proportions of T cells subsets differ significantly between blood and milk, particularly around calving. Corresponding variations in L-selectin expression may indicate a role for this molecule in regulating the movement of CD8+ and WC1+ T cells into the bovine mammary gland.     

Effect of Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine mammary gland lymphocytes

The aim of this study was to determine whether lymphocyte apoptosis is modulated by infections caused by Staphylococcus aureus and Streptococcus uberis. Samples of cell populations were obtained by lavage of the mammary glands at 4 intervals (24, 48, 72 and 168 h) following infection. The percentage of apoptotic lymphocytes peaked at 168 h after challenge with S. aureus or S. uberis. Subsequent experiments focused on in vitro cultivation of mammary gland lymphocytes with S. aureus and S. uberis. These experiments showed a lower percentage of apoptotic lymphocytes following 3 h of cultivating cells with bacteria than after cultivation without bacteria. The results demonstrate that during both experimental infection of bovine mammary glands with S. aureus or S. uberis and during in vitro cultivation of lymphocytes with S. aureus or S. uberis, apoptosis of lymphocytes is delayed.     

Effects of bovine leukemia virus infection on milk neutrophil function and the milk lymphocyte profile

The effects of bovine leukemia virus (BLV) on the immune response have been extensively investigated; however, its effects on mammary gland immunity are only speculative. Although BLV has a tropism for B cells, it can affect both adaptive and innate immunities because these systems share many effector mechanisms. This scenario is the basis of this investigation of the effects of BLV on mammary gland immunity, which is largely dependent upon neutrophilic functions. Thus, the present study sought to examine neutrophilic functions and the lymphocyte profile in the milk of naturally BLV-infected cows. The viability of the milk neutrophils and the percentage of milk neutrophils that produced reactive oxygen species (ROS) or phagocytosed Staphylococcus aureus were similar between BLV-infected and BLV-uninfected dairy cows. Furthermore, the expression of CD62L and CD11b by the milk neutrophils and the percentage of milk neutrophils (CH138⁺ cells) that were obtained from the udder quarters of the BLV-infected cows were not altered. Conversely, the median fluorescence intensity (MFI) representing intracellular ROS production and the phagocytosis of S. aureus, the expression of CD44 by the milk neutrophils and the percentage of apoptotic B cells were lower in the milk cells from BLV-infected dairy cows, particularly those from animals with persistent lymphocytosis (PL). The lymphocyte subsets were not different among the groups, with the exception of the percentage of CD5⁻/CD11b⁻ B cells, which was higher in the milk cells from BLV-infected cows, particularly those with PL. Thus, the present study provides novel insight into the implications of BLV infection for mammary gland immunity.     

Effect of intramammary infusion of beta-1,3-glucan or interleukin-2 on leukocyte subpopulations in mammary glands of sheep.

OBJECTIVE: To investigate effects of intramammary infusion of beta-1,3-glucan or recombinant ovine interleukin-2 (rOvIL-2) on blood and mammary leukocyte subpopulations and their expression of various surface antigens in sheep. ANIMALS: 12 healthy multiparous, nonpregnant, nonlactating fine-wool Merino ewes. PROCEDURE: Beta-1,3-glucan in pyrogen-free saline solution (PFSS; n = 6), rOvIL-2 in PFSS (3), or PFSS (3) was infused on days 0 and 7 in 1 udder half of each ewe. Jugular vein blood and mammary secretion samples were taken before infusion and on days 2, 7, and 21 after infusion. Total and differential leukocyte counts were obtained, and blood and mammary cells were labeled for flow cytometry. RESULTS: Slight swelling of the mammary glands was observed on day 2 after rOvIL-2 but not after beta-1,3-glucan infusion. Both substances induced significant increase in mammary secretion leukocyte numbers, compared with controls. Beta-1,3-Glucan induced an influx of monocyte/macrophages, whereas neutrophils were the predominating cell population after rOvIL-2 infusion. Beta-1,3-glucan induced selection for CD4+, B-cell+, WC1+, and L-selectin+ lymphocytes on day 14 after infusion. By comparison, rOvIL-2 induced selection for B-cell+ and L-selectin+ lymphocytes on days 14 and 21 and depletion of CD8 and, to some degree, of IL-2R+ lymphocytes. Beta-1,3-Glucan induced an increase in the proportion of CD14+ leukocytes, indicating selective migration of monocyte/ macrophages to the nonlactating udder. CONCLUSION: Beta-1,3-Glucan and rOvIL-2 can modulate nonspecific immunity in the udder of sheep but may exert their effects by differing mechanisms. Clinical Relevance-Stimulation of the nonspecific defense against udder infections may improve control of mastitis.