Correlation of Pasteurella haemolytica leukotoxin binding with susceptibility to intoxication of lymphoid cells from various species.

Pasteurella haemolytica, the causative agent of shipping fever pneumonia in cattle, produces a leukotoxin (LKT) which lyses ruminant leukocytes with high efficiency but is reputed to not affect leukocytes from nonruminant species. In this study, we tested the supposition that LKT binding correlates positively with susceptibility to intoxication of susceptible isolated bovine lymphocytes and lymphoma tissue culture cells (BL3 cells) and negatively with reputed nonsusceptible equine, porcine, and canine lymphocytes and human lymphoid tissue culture cells (Raji cells). Bovine lymphocytes and BL3 cells were highly susceptible to LKT intoxication, exhibiting both substantial increase in intracellular Ca(2+) concentration and marked leukolysis. Exposure of reputed LKT-nonsusceptible porcine lymphocytes and Raji cells to LKT caused a slightly increased intracellular Ca(2+) concentration but no leukolysis. No LKT effect was detected for equine and canine lymphocytes. LKT bound to lymphoid cells from all species tested. Intact 102-kDa LKT was recovered from exposed isolated lymphoid cell membranes. Pro-LKT acylation was not required for LKT binding to BL3 cells. LKT binding was rapid, with maximal binding occurring by 3 min, and was proportional to the LKT concentration in the range 0.04 to 4.0 microg/ml. For this LKT concentration range, BL3 cells bound more LKT than did porcine lymphocytes or Raji cells, suggesting that LKT binds to BL3 cells with higher affinity than to porcine lymphocytes or Raji cells. Above 4.0 microg/ml, LKT demonstrated saturable binding to BL3 cells. Neutralizing anti-LKT monoclonal antibody (MAb) MM601 diminished LKT binding to BL3 by 36% while decreasing leukolysis by 81%. In contrast, MM601 did not diminish LKT binding to Raji cells. Pretreatment of target cells with 120 microg of protease K per ml diminished LKT binding to BL3 cells by 75%, with only a 25% decrease in leukolysis. However, pretreatment with 150 microg of protease K per ml abolished the remaining 25% of LKT binding and 75% leukolysis. Therefore, P. haemolytica LKT binds rapidly to susceptible and to reputed nonsusceptible lymphoid cells. LKT binding resulting in species-specific leukolysis was characterized by high affinity, inhibition by MAb MM601, and relative resistance to protease K pretreatment of lymphoid cells. Two types of LKT binding to lymphoid cells are proposed. High-affinity binding leads to efficient leukolysis. In some lymphoid cells from reputed LKT-nonsusceptible species, low-affinity LKT binding may cause a low-efficiency increase in the intracellular Ca(2+) concentration without leading to leukolysis.

Role of intracellular calcium in Pasteurella haemolytica leukotoxin-induced bovine neutrophil leukotriene B4 production and plasma membrane damage.

Isolated neutrophils were used to study the intracellular calcium ([Ca2+]i) dependency of Pasteurella haemolytica leukotoxin-induced production of leukotriene B4 and plasma membrane damage. Exposure of neutrophils to leukotoxin caused a rapid and concentration-dependent increase in [Ca2+]i, followed by simultaneous plasma membrane damage and production of leukotriene B4. Removal of extracellular Ca2+, replacement of Ca2+ with other divalent cations, or exposure to high concentration of verapamil, an inhibitor of voltage-dependent calcium channels, inhibited leukotoxin-induced increases in [Ca2+]i, leukotriene B4 production, and membrane damage, thus indicating that influx of extracellular Ca2+ is necessary to produce these leukotoxin-induced neutrophil responses.

High-performance liquid chromatography of Pasteurella haemolytica leukotoxin using anion-exchange perfusion columns.

An exotoxin, called leukotoxin (LKT), from Pasteurella haemolytica, which had previously proved difficult to purify, was purified by high-performance liquid chromatography using rigid highly hydrophilic microparticulate anion-exchange columns. These anion-exchange stationary phases were employed to overcome difficulties of the relatively hydrophobic LKT interacting with dextran or styrene-based resins. While a short non-porous DEAE column allowed the partial microscale purification of the leukotoxin at pH 7.0, a high capacity strong anion-exchange column of the perfusion chromatography type permitted the purification of LKT on a much larger scale. The purification of the LKT on the large pore strong anion-exchange perfusion column was best achieved when three consecutive linear gradients at increasing NaCl concentration in 20 mM Tris buffer, pH 8.0, containing 6.0 M urea and 0.25% Tween 20 were used. Under these conditions, a better separation was obtained for the tetrameric and aggregate peaks of LKT from the early eluting contaminant peaks. This separation scheme allowed good recovery of activity and purification of the LKT to near homogeneity.

Giftedness: an exceptionality examined.

The study of giftedness has practical origins. High-level performance intrigues people. Theoretically, the study of giftedness is related to the psychology of individual differences and has focused on the constructs of intelligence, creativity, and motivation. At a practical level, the research is largely related to school and family contexts, which develop gifts and talents in children and youth. Although broadened definitions of giftedness have emerged, the most extensive body of research available for review concentrates on intellectual giftedness. The varying definitions of giftedness and the impact of social context and diversity on the development of talent pose significant challenges for the field. Finally, the study of exceptionally advanced performance provides insight into basic psychological processes and the school contexts that develop talents in children and youth.

Hemolytic activity of the Pasteurella haemolytica leukotoxin.

A Pasteurella haemolytica mutant incapable of producing leukotoxin was created by allelic replacement. Concentrated culture supernatants from wild-type P. haemolytica, but not from the mutant, contained the 102-kDa leukotoxin protein and lysed bovine lymphoma cells and sheep erythrocytes. Wild-type P. haemolytica demonstrated the typical beta-hemolytic phenotype on sheep and rabbit blood agar, whereas the mutant did not.

Chaotropic agents cause disaggregation and enhanced activity of Pasteurella haemolytica leukotoxin.

Pasteurella haemolytica biotype A, serotype 1 grown to late logarithmic growth phase in cell culture medium (RPMI 1640) produced highly aggregated leukotoxin. The multimer mass of the highly aggregated leukotoxin in 0.1 M sodium phosphate buffer pH 7.0 as determined by gel filtration chromatography on Sephacryl S400HR was approximately 8000 kDa. Resuspension of leukotoxin in phosphate buffer containing various chaotropic agents resulted in partial disaggregation of leukotoxin and enhanced leukotoxic activity. 3M guanidine disaggregated leukotoxin to a multimer mass of approximately 800 kDa and enhanced leukotoxic activity 3 to 20-fold. In 6 M urea or 1 M sodium thiocyanate, leukotoxin multimers were observed ranging in mass from 8000 kDa to 400 kDa, and activity enhancement was less than that for leukotoxin in 3 M guanidine. Several detergents were tested for enhancement of leukotoxic activity, but only 1% Tween 20 enhanced leukotoxic activity (4-fold), whereas 1.25% octylglucoside, 10 mM CHAPS, and 5 mM deoxycholate diminished and 1% Triton X-100 abolished leukotoxic activity.

Pasteurella haemolytica leukotoxin-induced synthesis of eicosanoids by bovine neutrophils in vitro.

Exposure of isolated bovine neutrophils to partially purified Pasteurella haemolytica leukotoxin caused increased synthesis of leukotriene B4 (LTB4) but not thromboxane B2 (TXB2) from endogenous arachidonic acid. Synthesis of LTB4 was closely correlated with leukotoxin-induced neutrophil lysis. At low toxin concentrations, LTB4 production lagged behind leukotoxin-induced neutrophil lysis over a 3-h period. The neutralizing monoclonal antileukotoxin antibody MM601 neutralized both leukotoxin-induced neutrophil lysis and LTB4 synthesis. Both leukotoxin-induced neutrophil lysis and LTB4 synthesis were Ca(2+)-dependent. When leukotoxin-induced LTB4 synthesis from exogenous arachidonic acid was examined, significant LTB4 synthesis occurred at 5 min of leukotoxin exposure, which was before leukotoxin-induced lysis developed. Leukotoxin-induced LTB4 synthesis from endogenous arachidonic acid appears to require leukotoxin-induced plasma membrane damage (occurring during neutrophil lysis), whereas LTB4 synthesis from exogenous arachidonic acid is initiated rapidly and occurs in the absence of plasma membrane damage.