Alterations in lipid metabolism induced by recombinant bovine tumor necrosis factor-alpha administration to dairy heifers

Endotoxin induces marked changes in lipid metabolism via its effects on cytokines. To evaluate the role of tumor necrosis factor-alpha (TNF) in mediating changes of lipid metabolism in ruminants, we performed a crossover saline-controlled study in Holstein heifers (n = 8; 394.0 kg average BW), investigating the metabolic effects of a single intravenous administration of recombinant bovine TNF (rbTNF, 5.0 microg/kg). Blood samples were taken from a jugular vein at 0 (1100, just before injection), 0.5, 6, 12, and 24 h after each treatment. Dry matter intake in the heifers was not affected by single administration of the rbTNF. The rbTNF produced early as well as later hypertriglyceridemia (P < 0.05) in dairy heifers. The rbTNF also induced an early and sustained rise (P < 0.05) in the plasma NEFA concentration. Plasma retinol concentration was decreased (P < 0.05) at 24 h after rbTNF injection, whereas the a-tocopherol concentration was not significantly affected by rbTNF treatment. At 0.5 and 24 h, there was an increase (P < 0.05) in the plasma concentration of the very-low-density lipoprotein (VLDL) fraction in rbTNF-treated heifers. Between 6 and 24 h after rbTNF treatment, concentration of the low-density lipoprotein fraction declined (P < 0.05) but the high-density lipoprotein fraction was not altered in the rbTNF-treated heifers. These results indicate that TNF produces a hypertriglyceridemic response associated with an increase of the VLDL fraction and a disturbance of retinol metabolism in dairy heifers.     

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.     

Effects of long-term administration of recombinant bovine tumor necrosis factor-alpha on glucose metabolism and growth hormone secretion in steers

OBJECTIVE: To investigate the effects of long-term administration of recombinant bovine tumor necrosis factor-alpha (rbTNF) on plasma glucose and growth hormone concentrations, and to determine whether treatment with rbTNF causes insulin resistance in steers. ANIMALS: 5 steers treated with rbTNF and 5 steers treated with saline (0.9% NaCl) solution (control). PROCEDURES: In experiment 1, rbTNF (5.0 microg/kg of body weight) or saline solution (5 ml) was administered SC daily for 12 days. Blood samples were obtained before treatment, and plasma was harvested for determination of glucose, insulin, and growth hormone (GH) concentrations. In experiment 2, insulin, glucose, or growth hormone-releasing hormone (GHRH) was administered IV on days 7, 9, and 11, respectively, after initiation of rbTNF or saline treatment in experiment 1. Plasma glucose and insulin concentrations were measured before and at various times for 4 hours after insulin or glucose administration. Plasma GH concentrations were measured at various times for 3 hours after GHRH administration. RESULTS: In experiment 1, administration of rbTNF resulted in hyperinsulinemia without hypoglycemia and decreased plasma GH concentrations. In experiment 2, plasma glucose concentrations were higher in steers treated with rbTNF and insulin than in controls. Plasma GH concentrations were lower in steers treated with rbTNF and GHRH than in controls. CONCLUSIONS AND CLINICAL RELEVANCE: Prolonged treatment with rbTNF induced insulin resistance and inhibited GHRH-stimulated release of GH in steers. Results indicate that rbTNF is a proximal mediator of insulin resistance and inhibits release of GH during periods of endotoxemia or infection.     

Administration of recombinant bovine tumor necrosis factor-alpha affects intermediary metabolism and insulin and growth hormone secretion in dairy heifers

Four experiments were conducted to clarify the effect of intravenous (i.v.) administration of recombinant bovine tumor necrosis factor alpha (rbTNF) on selected metabolites and on hormone secretion in Holstein heifers (n = 6; 347.0 kg average BW). In Exp. 1, rbTNF was injected at three dosage levels in a Latin square; 0 (CONT), 2.5 (TNF2.5), or 5.0 (TNF5) microg/kg BW. Plasma glucose and triglyceride concentrations were at first elevated (P < .05) by rbTNF treatment and then were decreased (P < .05) by TNF2.5 and TNF5. Plasma NEFA concentrations were increased (P < .05) in rbTNF-treated groups. The injection of rbTNF resulted in an increase in plasma insulin levels (P < .05 with TNF5) during the period between 2 and 24 h, except for the period between 6 and 8 h, after the treatment. In Exp. 2, 3, and 4, each heifer received i.v. injections of glucose (.625 mM/kg BW) + rbTNF (5 microg/kg) or glucose + saline (10 mL) (Exp. 2), insulin (0.2 U/kg) + rbTNF or insulin + saline (Exp. 3), and GHRH (0.25 microg/kg) + rbTNF or GHRH + saline (Exp. 4) at 1-wk intervals. In Exp. 2, rbTNF inhibited (P < .05) glucose-stimulated insulin secretion during the initial phase. Thereafter, plasma insulin was higher (P < .01) with the glucose + rbTNF treatment than with the glucose + saline treatment. Treatment with rbTNF inhibited the insulin-stimulated glucose utilization (Exp. 3) and GHRH-stimulated GH secretion (Exp. 4) during the initial phase. These results suggest that rbTNF directly and(or) indirectly affects the intermediary metabolism and hormone secretion in Holstein heifers.     

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.     

Release of tumor necrosis factor-alpha from bovine alveolar macrophages stimulated with bovine respiratory viruses and bacterial endotoxins

The release of tumor necrosis factor-alpha (TNF-) from cultured bovine alveolar macrophages (BAM) was evaluated following stimulation of BAM with bovine herpesvirus-1 (BHV-1), parainfluenza-3 (PI-3) virus, bovine respiratory syncytial virus (BRSV), Escherichia coli 0111:B4 endotoxin, Pasteurella haemolytica type 1 endotoxin, Pasteurella multocida endotoxin, and virus/endotoxin combinations. A cytotoxic assay system using Georgia bovine kidney cells as targets was used to measure TNF- activity. The cytotoxic activity was neutralized by an anti-human TNF- monoclonal antibody.

Stimulation of BAM with 1 median tissue culture infectious dose (TCID₅₀) of live or ultraviolet (UV)-inactivated PI-3 virus/cell resulted in release of TNF- in significantly (P<0.05) higher amounts than sham-induced BAM. The quantities of TNF- released after live or UV-inactivated BHV-1 or BRSV induction were not significantly higher than sham-induced BAM. E. coli 0111:B4, P. haemolytica type 1 and P. multocida endotoxins stimulated TNF- release in a dose-dependent manner. Sequential exposure of BAM to 1 TCID₅₀ per cell of either live BHV-1, PI-3 virus or BRSV and then 5 μg ml⁻¹ of either E. coli 0111:B4, P. haemolytica type 1 or P. multocida endotoxin caused a significant (P<0.05) reduction in detectable TNF- in seven of nine virus/endotoxin combinations tested, when compared with 5 μg ml⁻¹ of endotoxin alone. Parainfluenza-3 virus/endotoxin combinations stimulated higher TNF- release when compared with other virus/endotoxin combinations. Five out of six test animals had serum-neutralizing antibodies to PI-3 virus, one out of six had serum-neutralizing antibodies to BHV-1, and two out of six had serum-neutralizing antibodies to BRSV, suggesting a possible relationship between serum neutralizing antibodies and TNF- release from in vitro cultivated BAM.     

Effect of chronic administration of recombinant bovine tumor necrosis factor to cattle

Cachectin/tumor necrosis factor-alpha (TNF), a protein produced by macrophages upon stimulation, has been implicated as an important mediator of inflammatory processes and of clinical manifestations in chronic infectious diseases. In order to study further the potential role of TNF in infectious diseases, a homologous system was employed in which recombinant Escherichia coli (E. coli) derived bovine TNF (rBoTNF) was injected in cattle, either as a single bolus or in a repetitive treatment-regime. No clinical signs were observed, although changes occurred in hematologic and immunologic parameters when less than 0.5 mg of TNF/100 kg body weight was administered twice daily for 18 days. Prolonged treatment with 0.05-0.5 mg/100 kg induced histologic but no gross changes in the kidneys and liver. When doses were increased above 0.5 mg/100 kg, depression, anorexia, cachexia, and diarrhea appeared rapidly. Pathologic changes were apparent in various tissues including liver, kidneys, and lymphoid organs; body fat depots were depleted. Most of these changes appeared to be reversible; return to normal tissue-morphology occurred within 3 weeks of withdrawal of rBoTNF. The clinical and pathologic changes induced by prolonged rBoTNF administration resembled those observed in some chronic parasitic and viral infections of cattle in which macrophage-activation characteristically occur. Our finding may be relevant to the elucidation of the pathogenesis of these and other chronic infections.     

Expression of tumor necrosis factor-alpha in IgM+ B-cells from bovine leukemia virus-infected lymphocytotic sheep

Tumor necrosis factor (TNF)-alpha is thought to be one of the cytokines that account for bovine leukemia virus (BLV)-induced B-cell lymphoproliferative disorder, however, information on TNF-alpha expression in B-cells is limited. In this study, the expression of TNF-alpha in IgM(+) B-cells from BLV-infected sheep with or without lymphocytosis was determined. Freshly isolated IgM(+) B-cells from three sheep with lymphocytosis constitutively transcribed TNF-alpha mRNA. Although TNF-alpha mRNA expression in IgM(+) B-cells was transiently up-regulated after cell culture, TNF-alpha mRNA expression was markedly higher in lymphocytotic sheep when compared to that of non-lymphocytotic sheep or uninfected sheep. Expression of membrane-bound TNF-alpha on IgM(+) B-cells was also augmented in lymphocytotic sheep. TNF-alpha expression in lymphocytotic sheep may support the proliferation of B-cells.     

2''-5'' oligo-A-synthetase activity in bovine peripheral blood leukocytes and alveolar macrophages exposed to recombinant interferons and tumor necrosis factor-alpha.

In vitro treatment of bovine peripheral blood mononuclear leukocytes, polymorphonuclear neutrophilic granulocytes and alveolar macrophages with recombinant bovine interferons -alpha 1 1, -beta 2 or -gamma induced an immediate increase in the intracellular level of 2'-5' oligoadenylate synthetase activity. The induction was dose-dependent, with interferon -alpha 1 1 and -beta 2 being more potent than interferon-gamma. Maximal levels were reached within 10-12 h with IFN-alpha 1 1, which corresponded well with findings in vivo. In contrast to what has been found in nonlymphoid bovine cells, tumour necrosis factor-alpha did not potentiate the induction of 2-5A synthetase by interferons, neither did it by itself induce the enzyme.     

Effects of leptin and tumor necrosis factor-alpha on degranulation and superoxide production of polymorphonuclear neutrophils from Holstein cows

Leptin, a pleiotropic hormone regulating food intake and energy expenditure, has been shown to directly modulate human polymorphonuclear neutrophil (PMN) functions or indirectly through the action of tumor necrosis factor-alpha (TNF-alpha). Bovine PMN have considerable different characteristics from human PMN. For example, it does not respond to N-formyl-Methionyl-Leucyl-phenylalanine, a well known human PMN activator. In the present study, we tested the effects of leptin and TNF-alpha on superoxide production and degranulation of bovine peripheral PMN, in which both long isoform of leptin receptor (Ob-Rb) and TNF receptor 1 were expressed. Human leptin, human TNF-alpha, phorbol myristate acetate (PMA) and opsonized zymosan particles (OZP) did not stimulate degranulation responses, while zymosan-activated serum (ZAS) did. Neither leptin nor TNF-alpha enhanced the ZAS-induced degranulation responses. TNF-alpha, PMA, OZP and ZAS increased superoxide production in different magnitudes, whereas leptin did not. TNF-alpha, but not leptin, enhanced OZP- and ZAS-induced superoxide production, possibly, in part due to facilitating translocation of p47(phox), a component of NADPH oxidase. These results indicate that, unlike in human PMN, leptin does not have any direct effect on degranulation and superoxide production in bovine PMN, although TNF-alpha influences superoxide production.     

Increased pulmonary secretion of tumor necrosis factor-alpha in calves experimentally infected with bovine respiratory syncytial virus

Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory disease among calves in the Danish cattle industry. An experimental BRSV infection model was used to study the pathogenesis of the disease in calves. Broncho alveolar lung lavage (BAL) was performed on 28 Jersey calves, of which 23 were experimentally infected with BRSV and five were given a mock inoculum. The presence of the cytokine tumor necrosis factor alpha (TNF-alpha) in the BAL fluids was detected and quantified by a capture ELISA. TNF-alpha was detected in 21 of the infected animals. The amount of TNF-alpha in the BAL fluid of calves killed post inoculation day (PID) 2 and 4 was at the same very low level as in the uninfected control animals. Large amounts of TNF-alpha were detected on PID 6, maximum levels of TNF-alpha were reached on PID 7, and smaller amounts of TNF-alpha were seen on PID 8. The high levels of TNF-alpha appeared on the days where severe lung lesions and clinical signs were obvious and the amounts of BRSV-antigen were at their greatest. Although Pasteurellaceae were isolated from some of the BRSV-infected calves, calves treated with antibiotics before and through the whole period of the infection, as well as BRSV-infected calves free of bacteria reached the same level of TNF-alpha as animals from which bacteria were isolated from the lungs. It is concluded that significant quantities of TNF-alpha are produced in the lungs of the calves on PID 6-7 of BRSV infection. The involvement of TNF-alpha in the pathogenesis of, as well as the anti-viral immune response against, BRSV infection is discussed.     

Possible actions of tumor necrosis factor-alpha in ovarian function

Tumor necrosis factor-alpha (TNFalpha) is a multifunctional cytokine that was first described as a tumoricidal factor produced by activated macrophages. Extensive research over the last two decades has suggested that TNFalpha has physiologically diverse actions in ovarian function in a variety of species. TNFalpha and its specific receptors are present in the ovaries of many species. Furthermore, TNFalpha plays multiple and probably important roles in corpus luteum (CL) function as well as ovarian cell function throughout the estrous cycle. This review focuses on recent studies documenting TNFalpha in ovarian follicles and CL in several mammals. In addition, possible roles of TNFalpha in ovarian function throughout the estrous cycle and in the gestation period are discussed.     

Effect of recombinant bovine tumor necrosis factor-α on hormone release in lactating cows

Tumor necrosis factor (TNF)‐α is a powerful macrophage cytokine released during infection, circulating in the blood to produce diverse effects in the organism. We examined the effect of recombinant bovine TNF‐α (rbTNF‐α) administration on hormone release in dairy cows during early lactation. Twelve non‐pregnant Holstein cows were treated subcutaneously with rbTNF‐α (2.5 µg/kg) or saline twice (at 11.00 and 23.00 hours). At 11.00 hours the next day, the cows were given growth hormone‐releasing hormone (GHRH, 0.25 µg/kg), thyrotrophin‐releasing hormone (TRH, 1.0 µg/kg), thyroid‐stimulating hormone (TSH, 10 µg/kg) or adrenocorticotropic hormone (500 µg/head) via the jugular vein. In the growth hormone‐releasing hormone challenge, the plasma growth hormone concentration was lower in the rbTNF‐α group than in the control (saline) group. The growth hormone and TSH responses to TRH were also smaller in the rbTNF‐α group than in the control. The plasma prolactin response to TRH was not affected by the rbTNF‐α treatment. In the TSH challenge, the rbTNF‐α‐treated cows had lower responses, as measured by plasma triiodothyronine and thyroxine, than the control cows. The rbTNF‐α treatment produced an increase in the basal plasma cortisol level, but the cortisol response to adrenocorticotropic hormone was the same level in both groups. The plasma concentrations of TNF‐α and interleukin‐1β in the cows were elevated by the rbTNF‐α treatment. The milk yield was reduced by the rbTNF‐α administration during 4 days. These data demonstrate that TNF‐α alters the secretion of pituitary and thyroid hormones in lactating cows. This effect may contribute to the suppression of the lactogenic function of the mammary gland observed in cases of coliform mastitis with high circulating TNF‐α levels.     

Expression and characterization of a recombinant soluble form of bovine tumor necrosis factor receptor type I

A recombinant soluble bovine tumor necrosis factor receptor type I (sboTNF-RI) was expressed in the methylotrophic yeast Pichia pastoris and evaluated for its ability to inhibit bovine tumor necrosis factor alpha (TNF-alpha) cytotoxicity. A cDNA encoding the extracellular domain of bovine TNF-RI was placed under the control of the powerful and tightly regulated alcohol oxidase1 (AOX1) gene promoter of the pPICZa A vector and the resulting construct integrated into the 5' region of the alcohol oxidase genes of GS115 and KM71 strains of Pichia. Soluble bovine TNF-RI was secreted into the medium following induction of the AOX1 gene promoter with methanol, and purified to greater than 95% purity by ion-exchange chromatography. In in vitro assays, the purified recombinant sboTNF-RI will block the cytolytic activity of bovine TNF-alpha on WEHI 164 cells clone 13 by 50% when used at a concentration of 170 microg/ml, and by nearly 90% when used at a concentration of 310 microg/ml. Results of this study suggest that recombinant sboTNF-RI may have therapeutic value as a TNF inhibitor in cattle with coliform mastitis.     

Adrenocortical responses to tumor necrosis factor-alpha and interferon-gamma in cattle

The responses of plasma cortisol and adrenocorticotropic hormone (ACTH) were examined to intravenous injection of recombinant bovine tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (INF-gamma) in Holstein cows. INF-gamma induced dose-dependent rises in the plasma levels of both cortisol and ACTH, while TNF-alpha induced comparable plasma cortisol responses with much smaller rises in plasma ACTH. The results suggest a direct stimulatory action of TNF-alpha on cortisol secretion from the adrenal gland in cattle.     

Tumor necrosis factor-alpha enhances Haemophilus somnus lipooligosaccharide-induced apoptosis of bovine endothelial cells

Haemophilus somnus lipooligosaccharide (LOS)-induced apoptosis of bovine pulmonary artery endothelial cells has been shown previously to be dependent on capsase-8 activation. Activation of caspase-8 can occur via a death receptor-dependent mechanism (e.g., TNF- binding to TNF- receptor 1 (TNF-R1)). In this study, we tested the hypothesis that TNF- can enhance LOS-induced apoptosis of bovine endothelial cells. Addition of exogenous recombinant human TNF- alone failed to cause apoptosis, or enhance LOS-induced apoptosis, of bovine endothelial cells. However, blocking de novo protein synthesis by addition of cycloheximide significantly enhanced apoptosis of bovine endothelial cells by TNF-, LOS or TNF- and LOS in combination. Conversely, addition of soluble recombinant human (sTNF-R1) diminished LOS-induced apoptosis. Overall, these data suggest that LOS-mediated apoptosis may be due, in part, to activation of a TNR-R1-dependent death pathway.     

Commercially available antibodies to human tumour necrosis factor-alpha tested for cross-reactivity with ovine and bovine tumour necrosis factor-alpha using flow cytometric assays

A thorough understanding of the immune system, including the role of different cytokines, during inflammatory diseases in ruminants could lead to the development of new diagnostic methods and treatments. Tumour necrosis factor-alpha (TNF-alpha) is an important cytokine in the onset of the inflammatory responses. Unfortunately, the number of studies on cytokines, like TNF-alpha, in ruminants is limited due to a lack of species-specific reagents. As cytokines have remained rather conserved during evolution, cross-reactivity between animal species may occur. Therefore, the aim of the present study was to investigate 5 commercially available antibodies against human TNF-alpha for their ability to cross-react with ovine and/or bovine TNF-alpha, using a bead-based flow cytometric method. Two of the antibody clones (Mab 11 and 6401.1111) showed cross reactivity with ovine recombinant TNF-alpha in concentrations above 2.5 ng/ml. However, none of the antibodies detected TNF-alpha in bovine milk, or serum containing known concentrations of bovine TNF-alpha, as earlier determined with ELISA. The results could be due to inability of the antibodies to cross-react between species, but quenching of the signal by matrix proteins might also have lowered the response.     

Serum concentrations of tumor necrosis factor-alpha in neonatal calves with presumed septicemia

This study was performed to determine the concentrations of tumor necrosis factor (TNF) in the serum of neonatal calves with presumed sepsis and determine the correlation between serum concentrations of TNF and the severity and outcome of disease. Thirty-five sick calves < 30 days old that suffered from enteritis, respiratory disease, or both were considered suitable for inclusion in this study by satisfying clinical and laboratory criteria suggestive of septicemia. At admission, blood samples were collected from all calves to determine the prevalence of high concentrations of TNF. The clinical course and outcome of disease then were recorded. Of the 35 calves with presumed sepsis, 10 had high serum TNF concentrations. Scleral injection, weak or absent suckling reflex, sternal or lateral recumbency, unresponsive or comatose state, and death rate of calves with high serum TNF concentration were greater than those values for calves without high serum TNF concentration. Calves with high serum TNF concentration had significantly lower mean IgG (P < .001), globulin (P < .0001), and calcium (P < .0001) concentrations; greater serum creatinine concentrations (P < .0001); and > or = 2+ toxic changes in neutrophils than did calves without high serum TNF concentrations. Mean values for packed cell volume, band neutrophil count, and venous Pco2 were significantly (P < .007) higher in the group of calves with high serum TNF concentration. Results of this study indicate that serum TNF concentration is correlated with clinical criteria of sepsis in neonatal calves. A close association was apparent between disease severity and serum TNF concentrations in this group of calves with presumed septicemia.