Bovine Mammary Macrophages Research Articles

Reproducible isolation of bovine mammary macrophages for analysis of host pathogen interactions

BackgroundMacrophages residing in milk are vital during intramammary infections. This study sought to develop a method enabling the investigation of macrophage responses to pathogens. Streptococcus uberis is the predominant cause of bovine mastitis UK-wide and its pathogenesis is unusual compared to other intramammary pathogens. Previous studies utilise macrophage cell lines, isolated bovine blood derived monocytes, or macrophages from raw milk through complex or inconsistent strategies such as fluorescence activated cell sorting (FACS), centrifugation and selective adherence, and CD14 antibody-microbeads. The centrifuge steps required in the initial stages often damage cells. Thus, the aim of this study was to develop a reliable, reproducible, and cost-effective method for isolating mammary macrophages from milk in a way that allows their culture, challenge with bacteria, and measurement of their response ex-vivo.ResultsThis method achieves an average yield of 1.27 × 107 cells per litre of milk. Whole milk with somatic cell range of 45–65 cells/µL produced excellent yields, with efficient isolations accomplished with up to 150 cells/µL. This strategy uses milk diluted in PAE buffer to enable low-speed centrifugation steps followed by seeding on tissue-culture-treated plastic. Seeding 1,000,000 milk-extracted cells onto tissue culture plates was sufficient to obtain 50,000 macrophage. Isolated macrophage remained responsive to challenge, with the highest concentration of IL-1β measured by ELISA at 20 h after challenge with S. uberis. In this model, the optimal multiplicity of infection was found to be 50:1 bacteria:macrophage. No difference in IL-1β production was found between macrophages challenged with live or heat-killed S. uberis. Standardisation of the production of IL-1β to that obtained following macrophage stimulation with LPS allowed for comparisons between preparations.ConclusionsA cost-effective method, utilising low-speed centrifugation followed by adherence to plastic, was established to isolate bovine mammary macrophages from raw milk. This method was shown to be appropriate for bacterial challenge, therefore providing a cost-effective, ex-vivo, and non-invasive model of macrophage-pathogen interactions. The optimal multiplicity of infection for S. uberis challenge was demonstrated and a method for standardisation against LPS described which removes sample variation. This robust method enables, reproducible and reliable interrogation of critical pathogen-host interactions which occur in the mammary gland.

Priming from within: TLR2 dependent but receptor independent activation of the mammary macrophage inflammasome by Streptococcus uberis.

Streptococcus uberis is a member of the pyogenic cluster of Streptococcus commonly associated with intramammary infection and mastitis in dairy cattle. It is a poorly controlled globally endemic pathogen responsible for a significant cause of the disease worldwide. The ruminant mammary gland provides an atypical body niche in which immune cell surveillance occurs on both sides of the epithelial tissue. S. uberis does not cause disease in non-ruminant species and is an asymptomatic commensal in other body niches. S. uberis exploits the unusual niche of the mammary gland to initiate an innate response from bovine mammary macrophage (BMMO) present in the secretion (milk) in which it can resist the host immune responses. As a result - and unexpectedly - the host inflammatory response is a key step in the pathogenesis of S.uberis, without which colonisation is impaired. In contrast to other bacteria pathogenic to the bovine mammary gland, S. uberis does not elicit innate responses from epithelial tissues; initial recognition of infection is via macrophages within milk. We dissected the role of the bacterial protein SUB1154 in the inflammasome pathway using ex vivo bovine mammary macrophages isolated from milk, recombinant protein expression, and a panel of inhibitors, agonists, and antagonists. We combine this with reverse-transcription quantitative real-time PCR to investigate the mechanisms underlying SUB1154-mediated priming of the immune response. Here, we show that SUB1154 is responsible for priming the NLRP3 inflammasome in macrophages found in the mammary gland. Without SUB1154, IL-1β is not produced, and we were able to restore IL-1β responses to a sub1154 deletion S. uberis mutant using recombinant SUB1154. Surprisingly, only by blocking internalisation, or the cytoplasmic TIR domain of TLR2 were we able to block SUB1154-mediated priming. Together, our data unifies several contrasting past studies and provides new mechanistic understanding of potential early interactions between pyogenic streptococci and the host.

A Paradox in Bacterial Pathogenesis: Activation of the Local Macrophage Inflammasome Is Required for Virulence of Streptococcus uberis.

Streptococcus uberis is a common cause of intramammary infection and mastitis in dairy cattle. Unlike other mammary pathogens, S. uberis evades detection by mammary epithelial cells, and the host–pathogen interactions during early colonisation are poorly understood. Intramammary challenge of dairy cows with S. uberis (strain 0140J) or isogenic mutants lacking the surface-anchored serine protease, SUB1154, demonstrated that virulence was dependent on the presence and correct location of this protein. Unlike the wild-type strain, the mutant lacking SUB1154 failed to elicit IL-1β from ex vivo CD14+ cells obtained from milk (bovine mammary macrophages, BMM), but this response was reinstated by complementation with recombinant SUB1154; the protein in isolation elicited no response. Production of IL-1β was ablated in the presence of various inhibitors, indicating dependency on internalisation and activation of NLRP3 and caspase-1, consistent with inflammasome activation. Similar transcriptomic changes were detected in ex vivo BMM in response to the wild-type or the SUB1154 deletion mutant, consistent with S. uberis priming BMM, enabling the SUB1154 protein to activate inflammasome maturation in a transcriptionally independent manner. These data can be reconciled in a novel model of pathogenesis in which, paradoxically, early colonisation is dependent on the innate response to the initial infection.

Selenomethionine Suppressed TLR4/NF-κB Pathway by Activating Selenoprotein S to Alleviate ESBL Escherichia coli-Induced Inflammation in Bovine Mammary Epithelial Cells and Macrophages.

Inflammation is the hallmark of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli-induced bovine mastitis. Organic selenium can activate pivotal proteins in immune responses and regulate the immune system. The present study aimed to investigate whether selenomethionine (SeMet) attenuates ESBL E. coli-induced inflammation in bovine mammary epithelial cells (bMECs) and macrophages. Cells were treated with 0, 5/10, 10/20, 20/40, or 40/60 μM SeMet for 12 h and/or inoculated with ESBL-E. coli [multiplicity of infection (MOI) = 5] for 4/6 h, respectively. We assessed inflammatory responses, including selenoprotein S (SeS), Toll-like receptor 4 (TLR4), Ikappa-B (IκB), phospho-NF-κB p65 (Ser536), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and lactate dehydrogenase (LDH) activities. Treatment with 40/60 μM SeMet promoted cell viability and inhibited LDH activities in both bMECs and macrophages. Inoculation with ESBL-E. coli reduced cell viability, which was attenuated by SeMet treatment in bMECs and macrophages. SeMet increased ESBL E. coli-induced downregulation of SeS and decreased LDH activities, TLR4, IκB, phospho-NF-κB p65 (Ser536), IL-1β, and TNF-α protein expressions in bMECs and macrophages. In addition, knockdown of SeS promoted protein expression of TLR4-mediated nuclear factor-kappa (NF-κB) pathway and BAY 11-708 inhibited TNF-α and IL-1β protein levels in bMECs and macrophages after ESBL-E. coli treatment. Moreover, ESBL-E. coli inoculation increased monocyte chemoattractant protein 1 (MCP-1), C–C motif ligand 3 (CCL-3), and CCL-5 mRNA expressions in bMECs. In conclusion, ESBL-E. coli induced expression of MCP-1, CCL-3, and CCL-5 in bMECs and then recruited and activated macrophages, whereas SeMet attenuated ESBL E. coli-induced inflammation through activated SeS-mediated TLR4/NF-κB signaling pathway in bMECs and macrophages.

Sladek Z, Rysanek D: CD14 expression, apoptosis and necrosis in resident and inflammatory macrophages from virgin bovine mammary gland

This paper investigates the association between expression of CD14 and occurrence of apoptosis in blood monocytes, resident (<sub>RES</sub>MAC) and inflammatory macrophages from heifer mammary glands after infusion of PBS (<sub>INF</sub>MAC<sub>PBS</sub>) or LPS (<sub>INF</sub>MAC<sub>LPS</sub>). Resident macrophages (<sub>RES</sub>MAC) were obtained before, and inflammatory macrophages (<sub>INF</sub>MAC<sub>PBS </sub>and<sub> INF</sub>MAC<sub>LPS</sub>) 24 h after, induction of an inflammatory response using phosphate buffered saline (PBS) and lipopolysaccharide (LPS) in mammary glands of unbred heifers. Cell samples were analysed for differential counts, CD14 expression, apoptosis and necrosis using flow cytometry. In vitro cultivation led to a decrease in the proportion of living cells and to an increase in the proportion of apoptotic and necrotic cells in all macrophages and blood monocytes. In CD14<sup>+</sup> macrophages, the proportions of live cells increased and proportions of apoptotic and necrotic cells decreased after in vitro cultivation. We observed in CD14<sup>−</sup> macrophages and monocytes that the proportions of live cells decreased and proportions of apoptotic and necrotic cells increased after in vitro cultivation. Our experiments confirm that the expression of CD14 in bovine mammary gland macrophages and blood monocytes is associated with cell viability.  

Inhibition of bovine macrophage and polymorphonuclear leukocyte superoxide anion production by Haemophilus somnus

Virulent strains of the bovine opportunistic pathogen Haemophilus somnus ( Histophilus somni) cause multi-systemic diseases in cattle. One of the reported virulence factors that H. somnus may use to persist in the host is resistance to intracellular killing. We report here that H. somnus significantly ( P<0.001) inhibited production of superoxide anion (O 2 −) by bovine mammary and alveolar macrophages as well as by polymorphonuclear leukocytes. Inhibition of O 2 − was time- and dose-dependent and did not occur after incubation with Escherichia coli, H. influenzae, or Brucella abortus. Non-viable H. somnus, purified lipooligosaccharide, or cell-free supernatant from mid-log phase cultures did not inhibit O 2 − production, indicating that O 2 − inhibition required contact with live H. somnus. Furthermore, preincubation of phagocytic cells with cytochalasin B to prevent phagocytosis did not decrease the ability of H. somnus to inhibit O 2 − production. Some H. somnus isolates from the prepuce of healthy bulls were less capable or incapable of inhibiting macrophage O 2 − production compared to isolates tested from disease sites. Our results suggest that inhibition of O 2 − may be an important virulence factor exploited by pathogenic strains of H. somnus to resist killing by professional phagocytic cells.

Expression of CD3 and CD11b antigens on blood and mammary gland leukocytes and bacterial survival in milk of cows with experimentally induced Staphylococcus aureus mastitis

To differentiate early (1 to 8 days) from late (9 to 14 days) inflammatory phases and assess relationships between leukocyte phenotype and bacterial recovery in cows with Staphylococcus aureus-induced mastitis. 10 first-lactation Holstein cows. Blood and milk samples were collected from 4 or 6 cows before and after intramammary infusion of sterile broth or S. aureus, respectively. Flow cytometric expression of CD3 and CD11b antigens on blood and milk leukocytes, leukocyte differential counts, bacterial counts in milk, and somatic cell counts were determined longitudinally. Density of CD3 molecules decreased on blood lymphocytes and increased on milk lymphocytes after infusion of bacteria. Density of CD11b molecules on lymphocytes and phagocytes and percentage of CD11b+ lymphocytes in milk increased significantly after infusion; maximum values were achieved during the early inflammatory phase. Density of CD3 and CD11b molecules on milk lymphocytes and macrophages, respectively, 1 day after inoculation were negatively correlated with bacterial recovery on day 1 and days 9 to 14, respectively. Density of CD11b molecules on milk macrophages and the ratios of phagocyte to lymphocyte percentages and polymorphonuclear cell to macrophage percentages in milk differentiated the early from the late inflammatory phase. Activation of bovine mammary gland macrophages and T cells in response to intramammary infusion of S. aureus was associated with an inability to culture this bacterium from milk. Identification of specific inflammatory phases of S. aureus-induced mastitis in cows may allow for the design of more efficacious treatment and control programs.

Intracellular accumulation, subcellular distribution, and efflux of tilmicosin in bovine mammary, blood, and lung cells.

Tilmicosin is a semisynthetic macrolide antibiotic currently approved for veterinary use in cattle and swine to combat respiratory disease. Because the concentrations of tilmicosin are generally low in bovine serum, the interaction of tilmicosin with three types of bovine phagocytes (monocyte-macrophages, macrophages, and neutrophils from blood, lungs, and mammary gland, respectively) and mammary gland epithelial cells was evaluated to provide an understanding of potential clinical efficacy. After incubation with radiolabeled tilmicosin, uptake was determined and expressed as the ratio of the intracellular to the extracellular drug concentration. Accumulation of tilmicosin at 4 h of incubation by the alveolar macrophages (Cc/Ce 193) was 4 to 13 times more than that observed in monocyte-macrophages (Cc/Ce 43), neutrophils, (Cc/Ce 13), or mammary epithelial cells (Cc/Ce 20). Subcellular distribution showed that 70 to 80% of tilmicosin was localized in the lysosomes. Uptake in mammary gland cells was dependent on cell viability, temperature, and pH, but was not influenced by metabolic inhibitors or anaerobiosis. However, lipopolysaccharide exposure increased tilmicosin uptake by the bovine mammary macrophages and epithelial cells. When neutrophils and epithelial cells were incubated in the presence of tilmicosin and extracellular tilmicosin was then removed, 40% of the intracellular tilmicosin remained cell associated after 4 h of incubation (i.e., 60% effluxed), but only 25% remained in macrophages. These in vitro interactions of tilmicosin with bovine phagocytes and epithelial cells suggest an integral role in effecting clinical efficacy.

Phagocytosis of Streptococcus uberis by bovine mammary gland macrophages

The ability of macrophages isolated from the bovine mammary gland to phagocytose and kill Streptococcus uberis was investigated. The strains of S uberis used were selected on the basis of their known resistance (C197C) or sensitivity (EF20) to phagocytosis by bovine peripheral blood neutrophils in the same assay system. Macrophages isolated from mammary secretions collected during the mid-dry period were capable of phagocytosing both strains of S uberis in the presence of serum and skimmed milk. The removal of complement from serum by heat-inactivation did not influence the opsonization of S uberis for phagocytosis by mammary macrophages and both IgGI and IgG2 isotypes were found to opsonise both strains. The uptake of S uberis into the cells was confirmed by electron microscopy. Potential mechanisms by which S uberis could resist phagocytosis by neutrophils but not by macrophages are discussed.

Influence of Streptococcus dysgalactiae surface hydrophobicity on adherence to mammary epithelial cells and phagocytosis by mammary macrophages.

Bacterial surface hydrophobicity (SH) plays a role in adhesion of bacteria to host surfaces and ingestion by phagocytic cells. Streptococcus dysgalactiae (n = 60) isolated from bovine intramammary infections were examined for expression of SH after growth in Todd-Hewitt broth (THB) and THB supplemented with skim milk, whey, lactose, and casein. Strains were significantly more hydrophobic after growth in THB and THB plus whey and more hydrophilic after growth in THB plus skim milk. Both trypsin and proteinase K abolished SH in three strains tested. Mild pepsin treatment had little effect on SH, while heat treatment at 70 degrees or 80 degrees C abolished SH in two strains tested. A hydrophilic strain of S. dysgalactiae did not adhere as well to bovine mammary epithelial cells as a hydrophobic strain. Trypsin treatment significantly reduced adherence of a hydrophobic strain of S. dysgalactiae to epithelial cells while adherence of a hydrophilic strain remained unaltered. A hydrophilic strain of S. dysgalactiae was significantly more resistant to phagocytosis by bovine mammary gland macrophages than a hydrophobic strain. Differences in expression of SH may play an important role in determining the ability of S. dysgalactiae to establish successfully within the mammary gland.

Effects of in vitro supplementation of bovine mammary gland macrophages and peripheral blood lymphocytes with α-tocopherol and sodium selenite: implications for udder defences

Vitamin E and selenium have been observed to enhance the functions of bovine mammary gland macrophages and peripheral blood lymphocytes. In vitro supplementation with these compounds enhanced the production of neutrophil chemotaxins by macrophages stimulated with opsonised Staphylococcus aureus. Supplementation also enhanced the proliferation of peripheral blood lymphocytes in response to stimulation with concanavalin A but not to phytohaemagglutinin or pokeweed mitogen. There was no evidence of additive or synergistic effects of vitamin E and selenium. Results suggest that supplementation of cattle may optimise resistance to mastitis by enhancing the functions of resident macrophage and lymphocyte populations.

Phagocytosis of Streptococcus uberis by bovine mammary macrophages: opsonizing effect of bovine antiserum.

Five encapsulated and five non-encapsulated strains of Streptococcus uberis were tested for their ability to resist phagocytosis and intracellular killing by bovine mammary macrophages obtained during the non-lactating period. The percentage of phagocytosis and intracellular killing of encapsulated strains ranged from 43 to 51 and from 25 to 40, respectively. By contrast, the percentage of phagocytosis and intracellular killing of non-encapsulated strains ranged from 66 to 74 and from 65 to 75, respectively. Two encapsulated and two non-encapsulated strains were opsonized with homologous and heterologous pre-immune and immune bovine sera. Phagocytosis and intracellular killing of encapsulated strains increased significantly when opsonized with serum of cows vaccinated with a homologous and heterologous encapsulated strain. By contrast, no significant differences were observed when serum from cows vaccinated with a non-encapsulated strain was used. These results suggest that resistance to phagocytosis is a common feature among encapsulated strains of S. uberis, and that cross-reactivity exists between encapsulated strains.

Modulation of the intracellular survival of Brucella abortus by tuftsin and muramyl dipeptide

Tuftsin, a physiologic bioactive peptide of animal origin, and muramyl dipeptide, a synthetic bioactive glycopeptide of microbial origin, are known to enhance several recognized macrophage functions and increase non-specific resistance of the host against a number of pathogens. The influence of these two bioactive peptides was studied in permissive bovine mammary macrophages that were unable to control the intracellular replication of Brucella abortus and restrictive bovine mammary macrophages that were able to effectively reduce the intracellular survival of B. abortus. Addition of tuftsin (Thr-Lys-Pro-Arg) or muramyl dipeptide significantly ( P<0.03) enhanced the ability of the permissive macrophages to control the intracellular replication of B. abortus strain 2308 and resulted in the functional conversion of the permissive macrophages into restrictive macrophages. Addition of tripeptide tuftsin fragment (Lys-Pro-Arg), a natural inhibitor of tuftsin, to the medium completely abrogated the effect of tuftsin ( P<0.03). No additive effect on the ability of the macrophages to control the survival of B. abortus resulted from the combination of tuftsin and muramyl dipeptide.

Antiphagocytic effect of the capsule of Streptococcus uberis.

Non-opsonized encapsulated and non-encapsulated strains of Streptococcus uberis were incubated with bovine mammary macrophages and the percentage of phagocytosis and intracellular killing were determined. Seventy-seven percent of macrophages ingested non-encapsulated organisms with a killing rate of 75%. In contrast, 48% of macrophages ingested encapsulated bacteria with a killing rate of 35%. When strains were opsonized with homologous antiserum, differences were detected in the percentage of phagocytosis (84% vs. 48%) and intracellular killing (52% vs. 35%) of the encapsulated strain only. Effects were partially abolished when antiserum was absorbed with purified capsule, or when macrophages were pre-treated with purified capsular material. Electron microscopy of mammary macrophages incubated with the encapsulated strain of S. uberis showed the microorganism in contact with the macrophage cell membrane without signs of membrane activation. In contrast, the non-encapsulated strain induced formation of pseudopods and membrane ruffling. These results suggest that capsule may protect S. uberis from phagocytosis which may be due to a direct interaction of capsular material with macrophages.

Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection

Daily injections of an anti-inflammatory milk-derived factor (MDF) into mice increased resistance to Staphylococcus aureus challenge, and reduced leukocyte infiltration. Intraperitoneal injection of MDF into lactating mice prior to S. aureus intramammary challenge resulted in greater milk secretory activity and less inflammation compared with untreated controls, but had little effect on the number of S. aureus recovered from mammary tissue. Infusion of MDF directly into mouse mammary glands prior to challenge reduced S. aureus recovered after challenge. Incubation of bovine mammary macrophages in medium supplemented with MDF enhanced phagocytosis of opsonized S. aureus. In addition, infusion of 5 mg MDF into uninfected bovine mammary glands 24 h prior to S. aureus challenge resulted in fewer infections (five of ten) than in control quarters (seven of nine). Repeated daily injections of 5 mg MDF into S. aureus-infected quarters increased the percent of mammary neutrophils and decreased the recovery of S. aureus, but did not eliminate infections. Intravenous injection of 8 g MDF into cows resulted in pronounced leukopenia while the accompanying effect on mammary leukocytes was less marked but followed a similar course. Results suggest that the use of MDF in mice enhanced resistance to experimental infection and was beneficial in maintaining mammary secretory activity and reducing inflammation after bacterial challenge. In the cow, MDF promoted phagocytosis in vitro and was effective against challenge when infused intramammarily.

Function of bovine mammary macrophages as antigen-presenting cells

The ability of mammary macrophages treated with Staphylococcus aureus to induce antigen-specific T-cell proliferation was compared to that of the autologous blood monocytes. Induction of T-cell proliferation has been correlated with changes in major histocompatibility complex (MHC) class II antigen expression and interleukin 1 (IL-1) production by mammary macrophages and blood monocytes. The present study showed that both monocytes and mammary macrophages treated with S. aureus induced T-cell proliferation. However, there was a 3-fold decrease ( P<0.05) in T-cell proliferation in macrophage cultures compared to those of blood monocytes, when these cells were treated with S. aureus. Mammary macrophages, the cells less effective in stimulating T-cell proliferation, expressed lower levels (2-fold) of MHC class II molecules and produced less IL-1 (3-fold) than blood monocytes. These data suggest that S. aureus may affect macrophage-T cell interaction by modulating the expression of MHC class II molecules and the synthesis of IL-1 by macrophages.

Survival of smooth, rough and transposon mutant strains of Brucella abortus in bovine mammary macrophages

Transposon mutants offer a unique way to evaluate the role of lipopolysaccharide (LPS) by producing a theoretical single-gene difference between the original strain and the transposon mutant strain. Comparative survival of Brucella abortus smooth strain 2308, rough RB51, smooth strain 19, and two transposon mutant strains (rough strain 2308::Tn5 Lac Z [m106] and rough strain 19::Tn5 Lac Z [m3], was tested in restrictive bovine mammary macrophages that were able to effectively reduce the percentage of intracellular bacterial survival and permissive bovine mammary macrophages that were unable to control the intracellular replication of B. abortus. The theoretical single-gene difference between strain 19 and strain 19::Tn5 lac Z [m3] and between smooth virulent strain 2308 and rough transposon mutant 2308::Tn5 lacZ [m106] is likely related to differences in LPS content or structure. Significant ( P<0.05) reduction in the survival of rough strain 19::Tn5 Lac Z [m3] with no significant reduction in the rough transposon mutant strain 2308::Tn5 lacZ [m106] indicated that at least one factor other than LPS contributes to the intracellular survival of B. abortus in bovine macrophages.

Ability of mononuclear phagocytes from cattle naturally resistant or susceptible to brucellosis to control in vitro intracellular survival of Brucella abortus

The role of bovine mammary macrophages and peripheral blood monocyte-derived macrophages in natural resistance to bovine brucellosis was evaluated. A group of 11 naturally resistant and 10 chronically infected susceptible cows was studied following challenge with Brucella abortus. Macrophages from a greater proportion (P less than 0.026) of naturally resistant cows were significantly superior to macrophages from susceptible cows in their ability to inhibit the in vitro intracellular replication of B. abortus after challenge exposure. Studies of a second group of cows used mammary macrophages from 12 heifers and blood monocyte-derived macrophages from 22 bulls and heifers. These tests were completed before exposure to B. abortus, using mammary macrophages (P less than 0.039) and blood monocyte-derived macrophages (P less than 0.045), and also showed that macrophages from naturally resistant cattle were significantly superior in their ability to control the in vitro intracellular replication of B. abortus. Our data indicate that the mononuclear phagocytes from more than 80% of the resistant cattle controlled intracellular replication of B. abortus significantly better than did mononuclear phagocytes from susceptible cattle. Mononuclear phagocyte function appears to be an important factor in determining natural resistance to bovine brucellosis.

Ability of bovine mammary macrophages to enhance proliferation of autologous blood and mammary secretion lymphocytes

Cells were obtained by centrifuging the mammary secretion of healthy udders of 19 cows during the dry-period and during mid-lactation. The suspended cells were incubated in plastic wells. Those adhered cells classified as mammary macrophages were incubated with pokeweed mitogen (PWM). Autologous peripheral blood lymphocytes were added to wells containing untreated macrophage cultures or cultures pretreated with PWM. In seven cows autologous dry-period mammary lymphocytes were added instead of blood lymphocytes. The macrophages + lymphocyte cultures were subjected to the lymphocyte stimulation test (LST). For comparison, peripheral blood lymphocytes and dry-period secretion lymphocytes were also subjected to the LST in the presence of PWM. In all cases, mitogenic responses were higher in pretreated macrophage cultures than in background control cultures. The stimulation indices (SI) showed that PWM-pretreated dry-period mammary macrophages enhanced the proliferation of autologous peripheral blood lymphocytes to a greater extent than did blood lymphocytes plus PWM (49 +/- 10 v. 30 +/- 6; P less than or equal to 0.05). Mammary macrophages taken from the same cows but during midlactation also clearly induced proliferation of autologous peripheral blood lymphocytes but to a lesser extent than dry-period macrophages (16 +/- 2 v. 49 +/- 10; 16 +/- 2 v. 30 +/- 6; P less than or equal to 0.01 and P less than or equal to 0.05). The PWM pretreatment of mammary macrophages increased the proliferation of autologous dry-period mammary lymphocytes by at least a factor of three (28 +/- 8 v. 8 +/- 2 P less than or equal to 0.05). The present results indicate that bovine mammary macrophages pretreated with PWM enhance proliferation as well as modulation of mammary and peripheral blood lymphocytes. The modulation of lymphocyte stimulation as demonstrated here in vitro, has great significance regarding aspects of local immunostimulation related to modern treatment of mastitis.

Chemotactic factors for bovine neutrophils in relation to mastitis

The chemotactic effect of a variety of agents for bovine blood polymorphonuclear neutrophils (PMN) was evaluated in vitro in assays of migration under agarose. Activated serum and leukotriene B4 showed significant chemotactic activity whereas various bacterial products, formyl peptides, casein and platelet activating factor failed to attract bovine PMN. In vitro cultures of bovine mammary gland macrophages released chemotactic activity for homologous PMN when stimulated by addition of opsonised, killed Staphylococcus aureus, Escherichia coli or Zymosan or sterile E. coli culture filtrates or endotoxin. No significant activity was produced by unstimulated macrophages. Pharmacological levels of various inhibitors or arachidonic acid oxygenation had no significant effect on the generation of PMN chemoattractants by mammary macrophages.