Sex steroids and growth factors in the regulation of mammary gland proliferation, differentiation, and involution.

The mammary gland is subjected to major morphological and biochemical changes during the lactation cycle. It is therefore not surprising that this dynamic process is strictly controlled. The importance of the sex steroid hormones 17beta-estradiol and progesterone for normal development of the mammary gland was recognized several decades ago and has been unequivocally confirmed since. Furthermore, it is now also established that the influence of sex steroids is not restricted to mammogenesis, but that these hormones also control involution. Another important regulatory role is played by growth factors that have been shown to modulate survival (epidermal growth factor, amphiregulin, transforming growth factor alpha, insulin like growth factor, and tumor necrosis factor alpha) or apoptosis (tumor necrosis factor alpha, transforming growth factor beta) of mammary cells. However, the molecular mechanism underlying the influence of sex steroid hormones and/or growth factors on the development and function of the mammary gland remains largely unknown to date. Also scarce is information on the interaction between both groups of modulators. Nevertheless, based on the current indications compiled in this review, an important functional role for sex steroid hormones in the lactation cycle in co-operation with growth factors can be suggested.

Differential leukocyte count method for bovine low somatic cell count milk.

Whereas many differential leukocyte count methods for high somatic cell count (SCC) milk from mastitic cows are available, only a few have been developed for low SCC milk. We have developed a flow cytometric differential leukocyte count method for low SCC milk. The procedure consists of 1) 1.5 ml of diluted milk sample (30%, vol/vol dilution with PBS), 2) centrifugation, 3) leukocyte labeling with SYTO 13 and 4) flow cytometric analysis. Four major leukocyte populations can be clearly identified in the green fluorescence-side scatter dot plot: lymphocytes and monocytes (LM), polymorphonuclear neutrophils (PMN), mature macrophages (Mphi), and cells with apoptotic features based on chromatin condensation and nuclear fragmentation. The optimal processing temperature was 20 degrees C. Significant differences among samples with similar differential leukocyte counts were found. Storage of milk samples during 2 d at 7 degrees C had no effect on differential leukocyte count. Using the new method, differential leukocyte count was performed in low SCC milk samples from cows in early, mid, and late lactation. In accordance with previous studies, PMN and Mphi percentages were lower and LM percentages were higher in early lactation than in the other stages of lactation. The percentage of cells with apoptotic features was higher in early lactation than in mid and late lactation. In conclusion, a rapid, simple, accurate, and reproducible standard procedure was developed to determine the differential leukocyte count (Mphi, PMN, LM, and cells with apoptotic features) of bovine low SCC milk.

Effect of enrofloxacin treatment on plasma endotoxin during bovine Escherichia coli mastitis.

OBJECTIVE AND DESIGN: To investigate the effect of enrofloxacin on endotoxin resorption during bovine Escherichia coli mastitis. ANIMALS: 12 healthy early post partum Holstein cows. TREATMENT: Mastitis was induced by intramammary infusion of 10(4) cfu E. coli P4:032. Six cows were treated twice according to the usual enrofloxacin therapy: 5 mg/kg enrofloxacin 1) intravenously at 10 h and 2) subcutaneously at 30 h after challenge. The other 6 cows served as non-treated controls. METHODS: Blood and milk samples were collected at several time points after challenge. LPS in plasma was quantified using the limulus amoebocyte lysate (LAL) assay. The somatic cell count (SCC) and cfu of milk samples were also analysed. RESULTS: Occasional LPS peaks were detected in the plasma of 2 control cows at 6 h post-challenge and of 1 enrofloxacin-treated cow at 10 h post-challenge (P < 0.01 and P < 0.05, respectively, in comparison with time 0), just before enrofloxacin treatment. After enrofloxacin treatment, no significant LPS amounts were detected in the plasma of treated cows, but neither in the control cows. CONCLUSION: During induced coliform mastitis, LPS resorption in plasma occured only sporadically and within 10 h post-challenge. Whereas enrofloxacin treatment clearly limited bacterial growth in milk, significant effects on LPS resorption could not be detected. This suggests that enrofloxacin treatment of E. coli mastitis is predominantly beneficial by its bactericidal activity and is not associated with enhanced resorption of endotoxins.

Effect of isoproterenol and dexamethasone on the lipopolysaccharide induced expression of CD11b on bovine neutrophils.

The present experiments investigate the changes in expression of CD11b on bovine neutrophils and its modulation by isopropylnoradrenaline (IPN, isoproterenol), dexamethasone (DX), phenylephrine (alpha-agonist) and clenbuterol (beta-agonist). Both IPN and DX caused a dose-dependent inhibition of LPS-induced CD11b expression. A combination of IPN and DX elicited a synergistical decrease of the CD11b expression. Clenbuterol mimicked the effect of IPN, whereas phenylephrine did not. The effect of IPN and DX could at least partly be mediated through a decreased TNF-alpha production by monocytes since tumor necrosis factor-alpha (TNF-alpha) is shown to mediate a dose-dependent CD11b up-regulation. Stimulation of stress hormone receptors partly immuno-suppresses neutrophil functions by inhibition of CD11b expression on the neutrophil surface upon LPS stimulation. This inhibition is probably related to a decrease in TNF-alpha production. A similar mechanism of immuno-suppression could contribute to the higher susceptibility of cattle to Gram-negative bacterial infections of the udder and lung during periods of stress.

Immunological aspects of pregnancy-associated glycoproteins.

The incidence of severe cases of acute E. coli mastitis in dairy cows is highest during early lactation. This phenomenon has been associated with a decreased function and decreased numbers of circulating polymorphonuclear neutrophil leukocytes (PMN). The cause of this impaired function and decreased number is poorly understood. Stress, hormonal and metabolic alterations around parturition and the onset of lactation may play a role in this phenomenon. Several molecules, such as cortisol and beta-hydroxybutyrate have been found to alter the oxidative burst activity of circulating PMN around parturition. Pregnancy-Associated Glycoprotein (bPAG) could also be involved. The theory of immunosuppression by bPAG was investigated because analogous glycoproteins produced by the placenta of other species exert local immunosuppression in order to maintain the histoincompatible feto-maternal unit. The production and subsequent release into the maternal circulation of bPAG is ensured by the binucleate cells from the trophoblast and starts already at implantation. However, peak levels are only reached 1 week before parturition. Due to the long half-life time of this molecule, high levels are found in plasma until 2 weeks after calving. The co-occurrence of the impairment of PMN oxidative burst activity in the early postpartum period and a peak in plasma bPAG concentrations might support the hypothesis of an immunosuppressive effect of PAG. Moreover, an inhibitory effect of bPAG on the proliferation of bovine bone marrow progenitor cells has been found recently in our laboratory. bPAG occurs in colostrum, but its effect on milk cells has not been clarified. It is concluded that interaction between the physiology of reproduction and lactation on the one side and immune function on the other side in dairy cattle requires further research.

Flow cytometric analysis of the DNA content of bovine and human bone marrow cells.

The defence against infection in high-yielding dairy cows is correlated with the number and function of circulating neutrophils and depends on their production in bone marrow. Therefore, the DNA content of isolated bone marrow cell suspensions from 7 calves, 7 cows and 14 humans was assayed by flow cytometry. Bovine sternal bone marrow samples were collected within 30 min of death, and human marrow samples were collected by sternal puncture and aspiration. Mononucleated cells were isolated by gradient centrifugation. In the bone marrow samples from calves and cows, 35 +/- 2.6% and 31.8 +/- 1.5% of the isolated bone marrow cells respectively were in the S/G2/M-phase. The difference between calves and cows was not significant. In the human samples, only 12 +/- 0.8% of the cells were in the S/G2/M-phase. A significant (P < 0.001) difference was observed between the two species. These results indicated that the proliferative, in activity of haematopoietic cells is significantly higher in cattle than in humans.

Modulation of the inflammatory reaction and neutrophil defense of the bovine lactating mammary gland by growth hormone.

This review is focused on the possible interactions of prolactin and somatotrope hormone in the modulation of inflammation of the mammary gland. Several different models are examined: Escherichia coli, Streptococcus uberis, and endotoxin mastitis. Subsequently, the release of growth hormone and insulin-like growth factor during fever and mastitis, the immunophysiological effects of GH on E. coli mastitis, S. uberis and endotoxin mastitis, the galactopoietic action of rBST on healthy and mastitis cows as well as the immunologic effects of GH on leukocytes in healthy and diseased cows are discussed. It can be concluded that the underlying regulation of the neuro-endocrine network is fundamental in the normal function of the immune system.

Respiratory burst activity in activated and unstimulated isolated bovine blood neutrophils during experimentally induced Escherichia coli mastitis.

The respiratory burst activity, measured as H2O2 production, of isolated bovine polymorphonuclear leucocytes (PMN) was evaluated during experimentally induced Escherichia coli mastitis by means of flow cytometry in cells activated by phorbol 12-myristate 13-acetate (PMA) and in unstimulated cells. As expected, a significantly reduced respiratory burst activity was observed in PMA-activated PMN 18 h after intramammary inoculation with Escherichia coli. At this time only 75% of the PMA-activated PMN showed a respiratory burst, but with a higher intensity than that measured before and later after infection with Esch. coli. In addition, an increase in the respiratory burst activity was observed in unstimulated blood PMN during a short period at 18 h after infection, when up to 30% of the unstimulated PMN had a respiratory burst activity. The increase in the respiratory burst intensity of PMA-activated PMN and the spontaneously augmented production of reduced oxygen species by the unstimulated PMN during infection with Esch. coli might indicate the production of a natural stimulator of burst activity in circulation, most probably originating from the inflamed udder.

In vitro effect of ketone bodies, glucocorticosteroids and bovine pregnancy-associated glycoprotein on cultures of bone marrow progenitor cells of cows and calves.

Changes in the number, maturity and function of neutrophils, concomitant changes in plasma concentrations of hormones and metabolites, and the increased susceptibility of cows to infectious diseases around parturition, led us to investigate the effect of beta-hydroxybutyric acid (BHBA), acetoacetic acid (AcAc), hydrocortisone-21-acetate (HCAc) and bovine pregnancy-associated glycoprotein (bPAG) on the proliferation of bovine bone marrow progenitor cells in methylcellulose in vitro cultures. Myeloid progenitors were stimulated with concanavalin A-stimulated leukocyte conditioned medium (LCM) and erythroid progenitors with erythropoietin in the presence of hemin. Erythroid and myeloid colonies were scored after five and seven days, respectively. BHBA and AcAc induced inhibitory effects on the proliferation of bovine bone marrow cells at concentrations of 1.0, 2.5, and 5.0 mM. HCAc significantly inhibited growth of progenitors at concentrations of 10, 20, 50, and 100 ng/ml, and bPAG at concentrations of 2400 and 3000 ng/ml. The results of this study suggest that in the cow high concentrations of BHBA, AcAc, HCAc and bPAG, which can be reached in the circulation around calving, could alter the number of circulating neutrophils after parturition. This phenomenon might contribute to the increased susceptibility of dairy cows to environmental mastitis.

Effect of antibiotic induced bacterial clearance in the udder on L-selectin shedding of blood neutrophils in cows with Escherichia coli mastitis.

Bacterial clearance, L-selectin adhesion receptor expression and neutrophil recruitment during experimentally induced Escherichia coli mastitis were investigated. Acute mastitis was induced by injection of 10(4) cfu E. coli into both left quarters of 12 clinically healthy lactating cows, 2-6 weeks after parturition. Half the cows were treated intravenously 10 h after infection, and subcutaneously 30 h after infection with enrofloxacin. In both groups, bacterial count, milk serum albumin, milk somatic cell count, circulating leukocyte count and L-selectin expression on neutrophils were determined. Both groups responded to challenge with udder inflammation and fever. Treatment with enrofloxacin affected the elimination rate of bacteria at hours +14, +18, +24, +48, and +72 after challenge, but not the bacteriological cure rate (five out of six for treated and three out of six for nontreated cows). The drop in L-selectin expression found following challenge did not differ between groups throughout the study. No effect of treatment was found on SCC. Based on these findings, it may be concluded that when treatment is administered 10 h after infection, the number of bacteria in milk is not correlated with L-selectin expression on circulating PMN during experimentally induced E. coli mastitis. The initial bacterial load probably dictates the extent of the decrease in L-selectin expression and milk somatic cells.

Glucocorticosteroids and in vitro effects on chemiluminescence of isolated bovine blood granulocytes.

The effects of glucocorticosteroids on respiratory burst of bovine granulocytes were studied in vitro by means of (1) chemiluminescence (luminol-dependent, phorbol 12-myristate 13-acetate (PMA)-stimulated), (2) a cell-free chemiluminescence assay, and (3) a myeloperoxidase assay. Significant effects on cellular chemiluminescence were only observed at the highest, not obtainable in vivo, concentration for all drugs except betamethasone. Prednisolone induced inhibition at therapeutic doses. Also, flumethasone and dexamethasone induced significant inhibition at lower concentrations. In the cell-free assay, all glucocorticosteroids, except betamethasone, inhibited chemiluminescence at high concentrations. None of the glucocorticosteroids tested affected myeloperoxidase activity. The results indicated that the drugs do not affect NADPH-oxidase activity. The adverse effects may be due to scavenging of free oxygen radicals, or to interference with the interaction between luminol and the myeloperoxidase-H2O2-halide system. It can be concluded that most glucocorticosteroids show no adverse effects on the respiratory burst of bovine granulocytes in vitro at therapeutical concentrations.

Development and maturation of neutrophils.

Inflammatory processes require the activation of immunocompetent cells. In mammals, neutrophil polymorphonuclear leukocytes (PMN) constitute one of the essential body defences against diseases. In this article knowledge of the development and maturation of neutrophils, the control of haematopoiesis and of the factors that regulate neutrophil production is reviewed. As it has recently become apparent that neutrophils can be primed by cytokines to have enhanced functional activity, the future utilization of these growth factors in bovine immunotherapy is briefly discussed.

Triacylglycerol fatty acid composition of milk from periparturient cows during acute Escherichia coli mastitis.

Changes in fat concentration and triacylglycerol fatty acid (TGFA) composition were studied in milk from six periparturient cows 1 d before and 20 d after an experimentally induced Escherichia coli mastitis in the fore and rear homolateral quarters. Opposite fore and rear heterolateral quarters remained uninfected and were used as controls. Milk was collected from all individual quarters during the experiment. The fat concentration in milk from infected quarters did not change, but total fat production decreased owing to reduced milk production after the Esch. coli challenge. In milk from the heterolateral uninfected quarters fat concentration rose significantly 48 and 72 h after induction of mastitis, the rise being concomitant with a decrease in milk production. Throughout the experiment similar changes in TGFA composition were observed for both infected and uninfected quarters. There was an increase in all the even, odd-numbered, iso and anteiso short-chain TGFA from day +6 on after induction of mastitis. There was little change in the composition of 16:0 and 18:0 fatty acids, while the long-chain unsaturated fatty acids decreased. Using multivariate analysis, the results are presented visually. The observed changes in the TGFA can be ascribed to changes normally observed in cows' milk soon after parturition.

Classification of newly calved cows into moderate and severe responders to experimentally induced Escherichia coli mastitis.

In the present study newly calved cows were tentatively classified as moderate and severe responders to experimentally induced Escherichia coli mastitis based upon the reactive oxygen species (ROS)-generating capacity of their blood neutrophils before infection. The groups differed in blood and milk composition prior to infection. This initial classification was supported by the corresponding variation in clinical symptoms and in the changes in milk production and composition measured during mastitis. Responses of newly calved cows to Esch. coli challenge varied from mild to severe symptoms of inflammation in infected glands and differed in the intensity of systemic disturbances and general illness. Losses in milk yield and compositional changes were most pronounced in inflamed glands and in severe responders. In inflamed glands milk yield and composition did not return to preinfection level in either moderate or severe responders. The yields of lactose, alpha-lactalbumin, casein and fat followed the same pattern as milk yield. It is concluded that the severe and long lasting systemic disturbances observed in severe responders can be ascribed to absorption of endotoxin from infected glands into circulation, indicating the important role of endotoxin in the pathology of coliform mastitis in periparturient cows. Evaluation of the ROS-generating capacity of blood neutrophils and blood and milk composition before infection might help to predict the cow's sensitivity to Esch. coli mastitis.

Partial prostaglandin-mediated mechanism controlling the release of cortisol in plasma after intravenous administration of endotoxins.

In a first series of experiments we studied the influence of E. coli endotoxins or lipopolysaccharides (LPS) administered either intravenously (i.v.) or intramammarily (i.mam.) to lactating goats on plasma cortisol and rectal temperature (RT). Differences in the magnitude of the cortisol release and shape of the fever curve were observed. In both models maximal pyrexia and fever index (FI) were comparable. In a second series of experiments the influence of LPS on the plasma cortisol and RT was studied after i.v. injection of increasing doses of LPS:low (25 ng/kg), moderate (200 ng/kg) and relatively high (500 ng/kg). Although the cortisol response was dose dependent, the effect was not correlated with FI. The administration of flurbiprofen, a non steroidal antiinflammatory drug (NSAID), resulted in a complete inhibition of fever at all LPS doses and the cortisol release after administration of low doses LPS. This indicates a prostaglandin mediation. With moderate and high doses LPS the cortisol release was only partially inhibited and delayed suggesting a non prostaglandin mediated mechanism. In a third series of experiments the influence of flurbiprofen on fever and cortisol release was studied after i.mam. LPS administration. The observed increase of plasma cortisol and RT were completely abolished after flurbiprofen treatment. It is concluded that: 1) the increase of plasma cortisol after LPS administration in lactating goats is not related to hyperthermia per se, 2) the control of fever and cortisol release may, to some extent, differ according to the LPS dose and method of administration, 3) the cortisol release observed after moderate and high doses of LPS is probably controlled by two phenomena. The first being induced by cyclo-oxygenase metabolites, the second by intermediary mediators other than prostaglandins or by LPS itself. 4) Although an eight-fold higher dose of LPS was given i.mam., a cortisol release comparable to the lowest dose of LPS (25 ng/kg) was observed. These differences in cortisol release can be ascribed to 1) a detoxification of LPS at the level of the mammary gland or 2) a slower resorption of LPS from the mammary gland.

Role of prostaglandin-mediated mechanisms during experimentally induced endotoxin fever in the lactating goat.

The effects of endotoxin (LPS) on the cortisol, glucose, NEFA (non-esterified fatty acids), STH (somatotropin) and oxytocin levels in plasma of goats are described. The changes in plasma cortisol, STH and NEFA, as well as in RT (rectal temperature) were compared after i.v. and i.mam. administration of endotoxin. The other parameters, glucose and oxytocin, were followed only after i.v. endotoxin administration. The observed metabolic and hormonal alterations in plasma were also studied after pretreating the goats with the non-steroidal anti-inflammatory and antipyretic drug flurbiprofen in order to evaluate the possible involvement of prostaglandin in these phenomena. After i.v. administration of LPS a biphasic temperature curve for the highest dose of LPS with peak maxima at 1h and 4h after LPS challenge, was observed. Intramammary administration of endotoxin induces a monophasic fever response, with a latency time of approximately 3h, and peak values after 6h. The onset of the fever response in the i.v. experiments coincided with the oxytocin maximum and with early hyperglycemia. Intravenous endotoxin in goats also induces an increase in plasma NEFA, cortisol and STH. The early increase in NEFA, with a maximum after 2h and occurring before the fever peak, is followed by a significant rise in cortisol with peak effects after 3 h. The increase in plasma STH coincided with the decrease in plasma NEFA returning to control levels again. Peak concentrations in plasma STH occurred after 4 h. All the changes observed after the i.v. administration of endotoxin are dose-dependent. Pretreating goats with flurbiprofen completely abolished fever response, as well as the early hyperglycemia and the oxytocin release to i.v. LPS, indicating that these changes were prostaglandin-mediated and might be a reflexion of an activation of the sympathetic adrenomedullary system. The LPS-induced changes in plasma cortisol, NEFA and STH are only partly depressed and delayed by flurbiprofen. The residual hormonal responses to high doses of endotoxin suggest that an additional direct action of circulating endotoxins on the hypothalamus cannot be excluded. Intramammary LPS administration in goats only induced a very weak increase in plasma cortisol. The complex interplay of hormones and metabolic substances in the homeostasis of the inflammation reaction is discussed.

Effects of valerate and isobutyrate on fatty acid secretion by the isolated perfused mammary gland of the lactating goat.

The isolated mammary glands of six lactating goats were perfused with heparinized and oxygenated blood for 8 to 11 h. Adequate quantities of glucose, acetate and amino acids (including valine) were added to the perfusate. Either unlabelled valerate or unlabelled isobutyrate was added in excess to the perfusate of one gland, while the respective symmetrical gland was used as a control. After the administration of valerate, the proportions of the odd-numbered fatty acids (C11:0, C13:0, C15:0) in the milk fat, collected every hour during perfusion, rose progressively after 5 h until the end. The synthesis of milk fatty acids from valerate is discussed. After isobutyrate was added to the perfusate, isoC12:O, isoC14:0 and isoC16:0 in the milk fat increased as compared to the control. The effect of isobutyrate indicated that valine acted as a precursor of milk iso-branched fatty acids after its metabolisation to isobutyryl-CoA. During perfusion in the presence of the complete substrate mixture, the proportion of certain major milk fatty acids (C10:0, C12:0, C14:0 and C16:0) increased, whereas the proportion of C18:0 and C18:1 decreased. These effects have been ascribed to the presence of acetate and beta-hydroxybutyrate in the substrate mixture.

Changes in the fatty acid composition of goat milk fat after a 48-hour fast.

Five lactating goats were milked twice daily. After a control period of 3 days, they were fasted for 48 hr. The milk was collected at each milking. At the end of the fasting period, milk yield fell to 28% and milk fat to 55% of the original yield. The percentage of milk fat increased. Generally, the relative concentrations of fatty acids with less than or equal to 16 carbons, containing even and odd-numbered straight-chain as well as monomethyl-substituted fatty acids of the milk fat, decreased significantly 24 hr after food was withheld. The decrease was most pronounced for the fatty acids with the shortest chain lengths. Longer-chain acids increased or did not change. Iso and anteiso-acids seemed to follow the same, although less pronounced trend, the effect being obvious after 48 hr of fasting. It is suggested that the decline in the proportions of fatty acids with less than or equal to 16 carbons was due to the inhibition of mammary gland synthesis. The increase in the proportions of longer-chain fatty acids was ascribed to adipose tissue lipolysis.

Metabolism of leucine by the isolated perfused goat udder.

Seven lactating goat mammary glands from 6 goats were perfused for several hours in the presence of [U-14C]L-leucine (4 experiments) or [2-3H; 1-14C]DL-leucine (3 experiments) and received adequate quantities of glucose, acetate and amino acids. Radioactivity in casein was mainly recovered in leucine and 90% of casein leucine was derived from free plasma leucine. About 64% of the leucine molecules were used for casein synthesis. Up to 12% of the molecules were channelled into lipid synthesis, while the remaining (up to 24%) were metabolized to CO2. From the 3H/14C ratio of casein and casein leucine, it was calculated that 70-80% of the leucine molecules were reversibly transaminated before their incorporation into milk protein. However, only 4-8% of the plasma leucine molecules were transaminated during passage through the udder. Different pools for oxidation and for protein synthesis may be present in the goat mammary gland.

Propionate for fatty acid synthesis by the mammary gland of the lactating goat.

Isolated mammary glands of lactating goats were perfused with heparinized and oxygenated blood for 8 to 15 h. Adequate quantities of glucose, acetate, and amino acid were added to the perfusate. After addition of propionate to the perfusion blood, concentrations of odd-numbered and of monomethyl-substituted fatty acids other than those with iso and anteiso configuration increased in the milk fat. These acids seem to be synthesized de novo in the mammary gland. The increase of C17:0 concentration was weak and problematic. We suggest that propionate is acting as a precursor for monomethyl-substituted fatty acids by way of methylmalonyl-CoA. The activating effect of propionate administration upon milk fatty acid production was largest for odd-numbered followed by monomethyl-substituted fatty acids. No increase of iso acids was observed in milk fat in the propionate-infused glands whereas the increase of anteiso acids was extremely small. This agrees with the conception that iso and anteiso fatty acids are synthesized by rumen bacteria.