Cytotoxic tumor necrosis factor activity produced by equine alveolar macrophages: preliminary characterization.

Blood monocytes and alveolar macrophages (AM) were harvested from foals (aged 46 days to 6 months) and cultured in either medium alone or medium containing 10 micrograms/ml bacterial lipopolysaccharide (LPS). After 24 h, culture supernates were collected and analyzed for cytotoxic activity on sensitized L929 cells. Both monocytes and AM that had been treated with LPS produced significantly more cytotoxic activity than the same cell type exposed to medium lacking LPS. LPS-treated macrophages secreted significantly more cytotoxic activity (120 +/- 17.8 U/ml) than did LPS-treated monocytes (47.3 +/- 17.0 U/ml); however, constitutive production of cytotoxin by monocytes was higher (16.7 +/- 4.1 versus 1.2 +/- 1.2 U/ml). The identification of the cytotoxin as tumor necrosis factor (TNF) was strongly suggested by its reactivity with a rabbit antiserum directed against the N-terminal 15 amino acids of human TNF. TNF secretion by AM increased in a dose-dependent manner between LPS concentrations of 0.0001 and 1 microgram/ml, then leveled off. Most of the cytotoxic TNF activity produced by AM was secreted within the first 8 h after initial contact with LPS. Macrophage supernatant TNF was stable over a pH range of 6-11, but lost activity when kept at a pH less than 6. Equine TNF also was destroyed by exposure for 1 h to temperatures more than 60 degrees C. TNF bioactivity was recovered as a single peak after crude macrophage supernate was subjected to analysis by either anion exchange or gel filtration chromatography (molecular weight approximately 56,000).

Effects of polymyxin B and Salmonella typhimurium antiserum on horses given endotoxin intravenously.

Polymyxin B and an antiserum against an Re mutant Salmonella typhimurium were evaluated for protective effect in an equine model endotoxemia. Six 3- to 5-month-old foals were given endotoxin (0.25 micrograms/kg of body weight) IV after no pretreatment, or pretreatment with polymyxin B (6,000 U/kg, IV) or S typhimurium antiserum (1.5 ml/kg, IV). When given without pretreatment, endotoxin caused transient recumbency and increases in rectal temperature, and heart and respiratory rates. In addition, leukopenia and increases in circulating tumor necrosis factor (TNF) and interleukin 6 (IL-6) activities were detected. Compared with results obtained when endotoxin was given alone, pretreatment with polymyxin B resulted in significantly (P < 0.05) lower maximal plasma TNF and IL-6 activities, and significantly lower rectal temperature and respiratory rate. In contrast, compared with effects of endotoxin given without pretreatment, use of antiserum was associated with significantly (P < 0.05) higher respiratory rate, maximal plasma IL-6 activity, and total TNF response (as determined by areas under curves of plasma TNF vs time). These results indicate that polymyxin B may have potential as a treatment for equine endotoxemia. Salmonella typhimurium antiserum had no positive effect in this model, and, under certain conditions, may exacerbate the actions of endotoxin.

Tumor necrosis factor activity in the circulation of horses given endotoxin.

Serum and plasma from horses injected with endotoxin was examined for cytotoxic activity. Each of the cell lines, L929 and WEHI 164 clone 13, was sensitive to the cytotoxic effects of equine serum; however, a precipitation artifact caused by the use of isopropanol in the WEHI assay limited the use of this assay to samples containing less than 2 mg of protein/ml. In foals treated with a sublethal IV bolus of 5 micrograms of lipopolysaccharide (LPS)/kg and in adult horses given a low-dose continuous infusion of LPS (30 ng/kg/h for 4 hours), cytotoxic activity was detected in all serum or plasma samples taken between 30 minutes and 4 hours after LPS infusion began. In horses given either continuous or bolus LPS infusions, circulating cytotoxic activity peaked at 1 to 2 hours before decreasing sharply. The onset of pyrexia after LPS infusion coincided with the appearance of circulating cytotoxic activity, but the temperature remained high, even after cytotoxic activity disappeared. Treatment of horses with flunixin meglumine (1 mg/kg) appeared to blunt the pyrexic effect of low-dose continuous LPS infusion, but had no significant effect on circulating cytotoxic activity. Incubation of serum samples with an antibody raised against a portion of human tumor necrosis factor (TNF) resulted in the removal of greater than 90% of serum cytotoxicity, suggesting strongly that the cytotoxic activity was attributable to TNF. These findings are consistent with the hypothesis that TNF is an early acting mediator of the effects of endotoxin in the horse.

Effect of tumor necrosis factor antibody on synovial fluid cytokine activities in equine antebrachiocarpal joints injected with endotoxin.

Six horses received intra-articular injections of a mixture of 1 micrograms of endotoxin/5 mg of equine tumor necrosis factor (eqTNF) monoclonal antibody in 1 antebrachiocarpal joint and an equal volume (2 ml) of 1 micrograms of endotoxin/5 mg of control antibody in the opposite joint. Synovial fluid sample collection (1 ml) was accomplished by use of an indwelling, intra-articular catheter at postinjection hours (PIH) 0, 1, 1.5, 2, 5, and 8, and by arthrocentesis at PIH 24. Joint fluid samples were analyzed for nucleated cell count, protein concentration, and TNF, interleukin 6 (IL-6), IL-1, and IL-1-inhibitory activities. To monitor local inflammation, each carpus was graded semiquantitatively for swelling prior to each sample collection. Tumor necrosis factor, IL-1, or IL-1-inhibitory activity was not detected in any synovial fluid sample collected before endotoxin/antibody was administered. However, low IL-6 activity (< 100 U/ml) was found in 2 of 12 preinjection samples. In joints injected with endotoxin/control antibody mixture, maximal mean +/- SEM activities for TNF (1,019 +/- 310 U/ml), IL-1 (173 +/- 102 U/ml), and IL-6 (10.8 +/- 3.1 x 10(4) U/ml) were observed at PIH 2, 5, and 8, respectively. Tumor necrosis factor and IL-1 activities returned to baseline values by PIH 8 and 24, respectively; however, IL-6 activity remained high. Interleukin 1 inhibitory activity (27.4 +/- 2.25 IU/ml) was detected in all PIH-24 samples from control joints, but was not detected at any other time in control joints (limit of detection, 20 IU/ml). Tumor necrosis factor activity was not detected in any synovial fluid sample from joints treated with endotoxin/eqTNF antibody. In contrast, endotoxin IL-1 inhibitory activity (PIH 24) was higher in eqTNF antibody-treated joints (41.0 +/- 7.7 IU/ml) than in control joints, but the difference was not significant. Mean WBC count and protein concentration in control and treated joints were maximal at PIH 8. The curves for mean values of WBC count and total protein concentration were not significantly different in treated versus control joints. Swelling in each treated joint was either less than or the same as that in the opposite control joint at even, time in the initial 8 PIH. There was significant (P = 0.043) difference between treated and control joints at PIH 5 and 8. These results describe a profile of synovial fluid TNF, IL-1, IL-6 bioactivities, and IL-1-inhibitory activity during the initial 24 hours of synovitis induced by intra-articular administration of endotoxin in horses. Our eqTNF monoclonal antibody was effective in neutralizing TNF activity in synovial fluid when administered intra-articularly with endotoxin in horses. The induction of IL-1, IL-1 inhibitory activity IL-6, WBC, and total protein concentration responses are largely independent of TNF activity in synovial fluid of horses receiving endotoxin intra-articularly.

Secretion of tumor necrosis factor by endotoxin-treated equine mammary exudate macrophages: effect of dexamethasone and pentoxifylline.

Secretion of tumor necrosis factor (TNF) by equine mammary exudate macrophages (MEM phi) exposed to bacterial lipopolysaccharide (LPS) was dose-dependent and was maximal (216.5 +/- 51.9 U/ml) at 100 micrograms LPS/ml, the highest concentration tested. All concentrations of dexamethasone tested (10(-8) to 10(-4) M) significantly (P less than or equal to 0.05) inhibited TNF production by MEM phi when the agent was added 1 hour before LPS. Pretreatment with pentoxifylline at concentrations greater than 3 micrograms/ml also significantly (P less than or equal to 0.05) reduced secretion of TNF by MEM phi. The inhibitory effect of dexamethasone (10(-4) M) was observed when the agent was added to MEM phi from 30 minutes before until 4 hours after LPS. Pentoxifylline (100 micrograms/ml) significantly (P less than or equal to 0.05) suppressed TNF when added from 2 hours before until 2 hours after LPS; however, when pentoxifylline addition was delayed until 8 hours post-LPS, TNF production was enhanced. These apparent inhibitory effects of dexamethasone and pentoxifylline were not due to reduced macrophage viability or to interfering effects of the agents at the level of the TNF bioassay.