Influence of sex steroids on the viability and CD11b, CD18 and CD47 expression of blood neutrophils from dairy cows in the last month of gestation

In the period around parturition, cows experience an increased susceptibility for the development of Escherichia coli mastitis. This increased susceptibility has been correlated with a decreased functionality of neutrophils. In the current study, it is suggested that the decreased neutrophil functionality may be induced by the extensive alterations in sex steroid levels occurring around parturition. It was first hypothesized that 17beta-estradiol and progesterone influence the viability, apoptosis and necrosis of blood neutrophils from cows in their last month of gestation. Subsequently, it was hypothesized that 17beta-estradiol modulates the expression of CD11b, CD18 or CD47 thereby explaining its influence on the migration of bovine neutrophils. Neither 17beta-estradiol nor progesterone significantly influenced viability, apoptosis or necrosis in spontaneous apoptosis conditions. However, when apoptosis was induced with TNF-alpha and gliotoxin, progesterone exerted a survival effect (P<0.05). In addition, 17beta-estradiol treatment of bovine blood neutrophils significantly decreased the expression of CD47 (P<0.05) but not of CD11b or CD18. It can be concluded that 17beta-estradiol and progesterone do not affect spontaneous apoptosis of bovine blood neutrophils while a survival effect was observed for progesterone on induced neutrophils apoptosis. Moreover, our results concerning the influence of 17beta-estradiol on the CD11b, CD18 and CD47 expression extend previous demonstrations of the suppressive effect of 17beta-estradiol on neutrophils migration and indicate that the altered expression of CD47 may contribute to this phenomenon.     

Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells

Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkappaB activity in control and TNF-alpha treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kappaB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkappaB activity. As a surrogate marker of NFkappaB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-alpha enhanced NFkappaB activity in bovine mammary epithelial monolayers. p65 NFkappaB homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappaB consensus sequence, which specifically bound TNF-alpha inducible p50/p65 heterodimer. TNF-alpha induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-alpha induced IL-8 release. High dose interleukin-1beta (IL-1beta) induced IL-8 release, however significantly less potently than TNF-alpha. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-alpha or IL-1beta but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-alpha in activation of NFkappaB and release of IL-8 from bovine mammary epithelial cells.     

Phagocytosis and killing of Staphylococcus aureus by bovine neutrophils after priming by tumor necrosis factor-alpha and the des-arginine derivative of C5a

OBJECTIVES: To evaluate effects of proinflammatory mediators on phagocytosis and killing of Staphylococcus aureus, the oxidative burst (OB), and expression of receptors for opsonins by bovine neutrophils. SAMPLE POPULATION: Neutrophils from 10 cattle. PROCEDURE: Neutrophils were primed with recombinant bovine tumor necrosis factor-alpha (TNF-alpha) or the des-arginine derivative of bovine C5a (C5a(desArg)) and mixed with S aureus. Phagocytosis and OB were measured by use of flow cytometry. Rate of phagocytosis and intracellular killing were evaluated. Expression of receptors for immunoglobulins and the C3bi fragment of complement were estimated by use of flow cytometry. RESULTS: Priming of neutrophils by TNF-alpha improved phagocytosis of S aureus with a concentration-dependent effect. Phagocytosis of preopsonized washed bacteria was increased by activation of neutrophils with C5a(desArg). Phagocytosis was optimal when neutrophils primed with TNF-alpha were activated with C5a(desArg). The OB of phagocytizing neutrophils was highest when TNF-alpha and C5a(desArg) were used in combination. Bactericidal activity of neutrophils was stimulated by priming with TNF-alpha or C5a(desArg). Binding of bovine IgM or IgG2 to bovine neutrophils was not stimulated byTNF-alpha, C5a(desArg), or both, and aggregated IgG1 did not bind to neutrophils regardless of their activation state. Both TNF-alpha and C5a(desArg) increased expression of beta2 integrins (CD18), with the highest expression when they were used in combination. CONCLUSIONS AND CLINICAL RELEVANCE: The mediators TNF-alpha and C5a(desArg) stimulated phagocytic killing by neutrophils and potentiated each other when used at suboptimal concentrations. Bovine neutrophils have enhanced bactericidal activities at inflammatory sites when TNF-alpha, C5a(desArg), or both are produced locally.     

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.     

Rosette nanotubes inhibit bovine neutrophil chemotaxis

Migration of activated neutrophils that have prolonged lifespan into inflamed organs is an important component of host defense but also contributes to tissue damage and mortality. In this report, we used biologically-inspired RGD-tagged rosette nanotubes (RNT) to inhibit neutrophil chemotaxis. We hypothesize that RGD-RNT will block neutrophil migration through inhibition of MAPK. In this report, RNT conjugated to lysine (K–RNT) and arginine-glycine-aspartic acid-serine-lysine (RGDSK-RNT) were co-assembled in a molar ratio of 95/5. The effect of the resulting composite RNT (RGDSK/K–RNT) on neutrophil chemotaxis, cell signaling and apoptosis was then investigated. Exposure to RGDSK/K–RNT reduced bovine neutrophil migration when compared to the non-treated group (p < 0.001). Similar effect was seen following treatment with ERK1/2 or p38 MAPK inhibitors. Phosphorylation of the ERK1/2 and p38 MAPK was inhibited at 5 min by RGDSK/K–RNT (p < 0.05). The RGDSD/K-RNT did not affect the migration of neutrophils pre-treated with αvβ3 integrin antibody suggesting that both bind to the same receptor. RGDSK/K–RNT did not induce apoptosis in bovine neutrophils, which was suppressed by pre-exposing them to LPS (p < 0.001). We conclude that RGDSK/K–RNT inhibit phosphorylation of ERK1/2 and p38 MAPK and inhibit chemotaxis of bovine neutrophils.     

Expression and role of adhesion molecule CD18 on bovine neutrophils.

Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions.     

Activation-induced mobilization of secretory vesicles in bovine neutrophils.

OBJECTIVE: To characterize mobilization of secretory granules in bovine neutrophils. SAMPLE POPULATION: Neutrophils obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: Mobilization of secretory granules in bovine neutrophils was determined by measuring changes in cell-surface alkaline phosphatase activity on cells treated with various inflammatory mediators. Subcellular distribution of the alkaline phosphatase activity was determined by analysis of bovine neutrophil homogenates fractionated on density gradients. RESULTS: Alkaline phosphatase-containing secretory granules of bovine neutrophils were readily mobilized by a number of inflammatory agents, including platelet-activating factor, interleukin-8, tumor necrosis factor-alpha, lipopolysaccharide, leukotriene B4, and zymosan-activated plasma. In contrast, N-formyl-methionyl-leucyl-phenylalanine did not have a significant effect. Phorbol myristate acetate induced a biphasic response with up-regulation of cell-surface alkaline phosphatase at low doses and a return to baseline or even a reduction in cell-surface alkaline phosphatase at higher doses (> or = 10 ng/ml). Subcellular fractionation of bovine neutrophil homogenates revealed that alkaline phosphatase activity resided in light-density membrane vesicles (ie, location of secretory granules), which were distinct from specific, azurophil, and large granules. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine neutrophils respond to various inflammatory mediators by mobilizing alkaline phosphatase-containing secretory granules. This suggests that the process is an important early step in the host-defense response of bovine neutrophils.     

Direct stimulation of the oxidative activity of isolated equine neutrophils by TNF-alpha and IL-1beta

The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 x 10(6) cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017-167 ng ml(-1)) for 1.25 x 10(6) neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.     

Involvement of reactive oxygen species in TNF-alpha mediated activation of the transcription factor NF-kappaB in canine dermal fibroblasts.

The cytokine tumor necrosis factor-alpha (TNF-alpha) plays a major role in inflammatory and immune-pathological reactions of the skin. With respect to a possible therapeutic modulation of TNF-alpha mediated activation of Nuclear Factor-kappa B (NF-kappaB) in canine cutaneous inflammation, we investigated the role of NF-kappaB and the involvement of reactive oxygen species (ROS) in the TNF-alpha signalling pathway in dermal fibroblasts of the dog. TNF-alpha treatment resulted in the activation of NF-kappaB as assessed by electrophoretic mobility shift assay (EMSA). Additionally, NF-kappaB translocation was induced with butylhydroperoxide and antimycin A, but not with hydrogen peroxide. TNF-alpha stimulated NF-kappaB activation was partially inhibited by preincubation with the antioxidants alpha-lipoic acid and butylated hydroxyanisol (BHA). No superoxide generation following TNF-alpha stimulation could be detected in the supernatant of canine fibroblasts with the superoxide dismutase-inhibitable cytochrome c reduction test. In contrast, production of TNF-alpha dependent intracellular hydrogen peroxide, the dismutation product of the superoxide radical, was demonstrated spectroscopically by formation of electron dense cerium-hydroperoxide precipitates. With electron energy loss spectroscopy (EELS) significant cerium deposits were detected in the mitochondria, the endoplasmatic reticulum, the cytosol and to a lesser extent on the plasma membrane of canine fibroblasts indicating multiple hydrogen peroxide production sites. Peroxides, therefore, possibly play an important part in the redox-sensitive pathway of TNF-alpha dependent NF-kappaB activation in canine skin. An adjunctive therapy with appropriate antioxidants modulating NF-kappaB overactivation in cutaneous inflammation in the dog is promising.     

Aucubin ameliorates the LPS-induced inflammatory response in bovine endometrial epithelial cells by inhibiting NF-κB and activating the Keap1/Nrf2 signalling pathway

Cows are susceptible to pathogenic bacterial infection after pregnancy, leading to inflammation of the endometrium. Aucubin (AU) has been proven to exhibit highly effective anti-inflammatory activity, but its ability to protect against endometritis in dairy cows remains unclear. Therefore, the goal of the present study was to evaluate the protective effect of AU on the LPS-induced inflammatory response of bovine endometrial epithelial cells (BEECs). After pre-treating BEECs with AU (10, 20 and 50 μM) for 6 hr, the cells were stimulated with LPS for 3 hr. Subsequently, BEECs apoptosis was analysed by flow cytometry, the expression of pro-inflammatory cytokine mRNA was detected by qRT-PCR, and changes in NF-κB and Keap1/Nrf2 signalling were analysed by western blotting and immunofluorescence analyses. The results showed that AU can reduce TNF-α, IL-1β, IL-6, COX-2 and iNOS mRNA expression in BEECs and reduce cell apoptosis. Furthermore, AU significantly reduced the level of NF-κB p65 and IκB phosphorylation and inhibited the nuclear translocation of NF-κB p65. AU also activated the Keap1/Nrf2 pathway, promoting the nuclear transfer of Nrf2 and increasing Keap1, Nrf2, HO-1 and NQO1 mRNA and protein levels. Taken together, these results indicate that AU ameliorates the LPS-induced inflammatory response by inhibiting NF-κB and activating the Keap1/Nrf2 signalling pathway, which has a protective effect on BEECs.     

Bovine antibody dependent cell-mediated cytotoxicity (ADCC) effector cells

ADCC effector cells from bovine blood were separated by centrifugation, adherence and rosetting techniques. Each enriched cell population, peripheral blood mononuclear cells (PBM), null lymphocytes, monocytes and neutrophils, was then examined for its capacity to mediate ADCC. Utilizing heterologous sensitizing antisera it was found that monocytes had approximately twice the ADCC activity of null lymphocytes and that neutrophils had essentially no activity. However, when homologous sensitizing antisera were used it was found that neutrophils possessed the greatest activity followed by monocytes and null cells. Results confirm the existence of an ADCC active null lymphocyte in the bovine.     

Costimulatory effects of complement receptor type 3 and Fc receptor for IgG (FcgammaR) on superoxide production and signal transduction in bovine neutrophils

The present study evaluated the costimulatory effects of complement receptor type 3 (CR3) and Fc receptor for IgG (FcgammaR) on superoxide production and intracellular signal transduction in bovine neutrophils. Stimulation with opsonized zymosan (OPZ) and heat-aggregated bovine IgG (Agg-IgG) resulted in much greater superoxide production and chemiluminescent (CL) responses in normal neutrophils compared with those stimulated with OPZ or Agg-IgG only. Superoxide production and CL response were closely associated with the stimulant-induced rise of the intracellular calcium ([Ca2+]i) concentration, amount of tyrosine phosphorylated 100 kDa protein, and activation of p38 mitogen-activated protein kinase (p38 MAPK). No costimulatory effect was found for these receptors on superoxide production in CR3-deficient neutrophils. Costimulation of CR3 and FcgammaR on bovine neutrophils leads to enhancement of superoxide production and their signaling pathways and appears to be associated with enhancement of neutrophil functions.     

Antibacterial activity of bovine lactoferrin hydrolysate against mastitis pathogens and its effect on superoxide production of bovine neutrophils

Antibacterial activity of bovine lactoferrin hydrolysates (LFH) on microorganisms isolated from bovine mastitis, and superoxide (O(2)(-)) production of bovine neutrophils were evaluated. Antibacterial effects of LFH were measured in vitro against Staphylococcus aureus, coagulase-negative staphylococci, Streptococci, Enterococci, Escherichia coli, Klebsiella pneumoniae, yeast-like fungi and Prototheca zopfii isolated from clinical cases of bovine mastitis. To compare susceptibilities against LFH, minimal inhibitory concentration (MIC) values were determined by a micro-plate assay method. Most organisms were sensitive to LFH. Prototheca zopfii was highly sensitive to LFH; the growth of the microorganism was inhibited completely even at 1 mug/ml. Staphylococcus aureus and Escherichia coli were resistant to LFH. The production of O(2)(-) by bovine neutrophils was used to evaluate the effect of LFH administration on functional activity. Increase in O(2)(-) production by bovine neutrophils occurred upon addition of LFH to neutrophils. These results demonstrate that LFH possesses antibacterial activity against pathogens that cause mastitis and activates neutrophil superoxide production.     

p65 Homodimer activity in distal airway cells determines lung dysfunction in equine heaves

Nuclear factor-κB (NF-κB) activity, which is a key regulator of inflammatory gene expression, is increased in bronchial epithelial cells from horses suffering from heaves (a hypersensitivity-associated inflammatory condition of the lung). To determine whether this increased activity extends to distal airways and to other pulmonary cells, cells recovered by broncho-alveolar lavage (BAL) in healthy and heaves-affected horses were assessed for NF-κB activity. NF-κB activity was much higher in BAL cells from heaves-affected horses, especially during crisis (disease exacerbation), than in cells from healthy horses. Moreover, the level of NF-κB activity found in BAL cells was positively correlated to total lung resistance and to the proportion of neutrophils present in BAL fluid. Finally, prototypical p65–p50 NF-κB heterodimers were absent from BAL cells, which mostly contained p65 homodimers. These results (1) show that increased NF-κB activity is a general feature of heaves lung; (2) demonstrate the importance of p65 homodimers in neutrophilic inflammation; and (3) suggest that the use of specific NF-κB inhibitors could improve lung function in heaves-affected horses.     

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.     

Role of inflammatory mediators in priming, activation, and deformability of bovine neutrophils

OBJECTIVE: To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils. SAMPLE POPULATION: Blood collected from 5 healthy adult Holstein cows. PROCEDURE: Isolated neutrophils or whole blood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured. RESULTS: Incubation with TNF-alpha, IL-8, PAF, and LPS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. Incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and selectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.     

Generation of neutrophil chemoattractants by phagocytosing bovine mammary macrophages

Macrophages from bovine mammary gland were cultured in vitro and the growth medium collected at intervals. Using an in vitro system in which neutrophils migrated under agarose, both chemotactic and chemokinetic activity for bovine neutrophils was detected in supernatants of macrophage cultures to which heat killed preopsonised Staphylococcus aureus had been added. The suspensions of killed bacteria were not themselves chemotactic for neutrophils and no chemotactic activity was present either in supernatants from unstimulated macrophage cultures or in sonicated macrophages. The chemotaxin(s) was generated within two hours of the addition of staphylococci to the cultures and was largely stable to heating at 56 degrees C for 45 minutes, although its activity was reduced by boiling for 15 minutes. Traces of proteolytic activity were also detected in some supernatants. Substantial proteolytic activity was found in lysates of neutrophils. Unlike chemotaxis, proteolytic activity was suppressed by addition of milk from early lactation and containing high natural levels of protease inhibitors. Proteolytic activity was destroyed by boiling for 15 minutes.