Lafleur R L, Malazdrewich C, Jeyaseelan S, Bleifield E, Abrahamsen M S, Maheswaran S K
Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108, USA.
Microb Pathog. 2001 Jun;30(6):347-57. doi: 10.1006/mpat.2000.0438.
Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) and lipopolysaccharide (LPS) are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies have characterized in vitro responses of bovine alveolar macrophages (AMs) to Lkt and LPS. Activation of AMs with Lkt or LPS causes induction of proinflammatory cytokines, and Lkt causes cytolysis of AMs at higher concentrations. Since AMs are exposed to both of these bacterial virulence factors during disease, previous studies may have underestimated the possibility of functional interactions between Lkt and LPS. The purpose of this study was to characterize the effect of simultaneous exposure to both Lkt and LPS on AM cytolysis and proinflammatory cytokine expression. Using cellular leakage of lactate dehydrogenase as an indirect measure of cytolysis, we studied AM responses to Lkt alone, LPS alone and Lkt+LPS. We found that 80-200 pg/ml LPS, which does not itself cause cytolysis, synergistically enhanced the cytolysis induced by 2-5 Lkt units (LU)/ml Lkt. Northern blot analysis demonstrated that synergism between Lkt and LPS resulted in increased levels of IL-8 mRNA, and that the kinetic patterns of TNF-alpha and IL-8 mRNA expression induced by Lkt+LPS differed from those induced by each agent separately. Finally, the WEHI 164 (clone 13) bioassay was used to show that Lkt/LPS synergism resulted in enhanced secretion of biologically active TNF-alpha. These results provide direct evidence of synergism between Lkt and LPS in AM cytolysis and inflammatory cytokine expression. Additional studies to characterize the molecular basis of this phenomenon are indicated.
溶血曼氏杆菌（巴斯德氏菌属）白细胞毒素（Lkt）和脂多糖（LPS）是导致牛肺炎性巴氏杆菌病肺损伤发病机制的主要毒力因子。先前的研究已对牛肺泡巨噬细胞（AMs）对Lkt和LPS的体外反应进行了表征。用Lkt或LPS激活AMs会导致促炎细胞因子的诱导，并且Lkt在较高浓度下会导致AMs的细胞溶解。由于在疾病过程中AMs会同时接触这两种细菌毒力因子，因此先前的研究可能低估了Lkt和LPS之间功能相互作用的可能性。本研究的目的是表征同时暴露于Lkt和LPS对AMs细胞溶解和促炎细胞因子表达的影响。我们使用乳酸脱氢酶的细胞泄漏作为细胞溶解的间接指标，研究了AMs对单独的Lkt、单独的LPS以及Lkt+LPS的反应。我们发现，本身不会引起细胞溶解的80 - 200 pg/ml LPS会协同增强由2 - 5 Lkt单位（LU）/ml Lkt诱导的细胞溶解。Northern印迹分析表明，Lkt和LPS之间的协同作用导致IL - 8 mRNA水平升高，并且Lkt+LPS诱导的TNF - α和IL - 8 mRNA表达的动力学模式与单独由每种试剂诱导的模式不同。最后，使用WEHI 164（克隆13）生物测定法表明，Lkt/LPS协同作用导致生物活性TNF - α的分泌增加。这些结果提供了Lkt和LPS在AMs细胞溶解和炎性细胞因子表达中协同作用的直接证据。表明需要进行额外的研究来表征这种现象的分子基础。
