The biphasic mRNA expression pattern of bovine interleukin-8 in Pasteurella haemolytica lipopolysaccharide-stimulated alveolar macrophages is primarily due to tumor necrosis factor alpha

Abstract

Pasteurella haemolytica serotype 1 is the bacterial agent responsible for the pathophysiological events associated with bovine pneumonic pasteurellosis. Our previous studies support a role for the lipopolysaccharide (LPS) from P. haemolytica in the induction of proinflammatory cytokines. One of the pathological hallmarks of bovine pneumonic pasteurellosis is an influx of neutrophils into the alveolar spaces. This pronounced influx suggests the local production of a chemotactic factor(s) such as interleukin-8 (IL-8). In the context of the lung, the alveolar macrophage appears to be the major producer of IL-8, a proinflammatory cytokine with potent neutrophil chemotactic activity. By using Northern blot analysis, we have examined the kinetics of IL-8 mRNA expression in P. haemolytica LPS-stimulated bovine alveolar macrophages and found that 1 ng of LPS per ml induces maximal expression of IL-8 mRNA. The results also indicate a biphasic time course expression pattern in which IL-8 mRNA levels peak between 1 and 2 h in the first phase and between 16 and 24 h in the second phase (P < 0.01). In addition, monospecific polyclonal antibodies were used to demonstrate the role of tumor necrosis factor alpha (TNF-alpha) in the second phase of IL-8 mRNA expression. Our findings support a role for P. haemolytica LPS and TNF-alpha in the induction of IL-8 from bovine alveolar macrophages.

FIG. 1

Length of time required by bovine AMs to become quiescent for bovine IL-8 mRNA. (A) Northern blot analysis with the IL-8 probe was performed as described in Materials and Methods. (B) Relative levels of hybridization signal were normalized to GAPDH. The data shown are representative of two separate experiments.

FIG. 2

Bovine IL-8 mRNA expression in bovine AMs stimulated with various concentrations of P. haemolytica LPS for 1 h. (A) Northern blot analysis with the IL-8 probe was performed as described in Materials and Methods. (B) Relative levels of IL-8 mRNA were normalized to GAPDH mRNA levels. The data shown are representative of three separate experiments.

FIG. 3

Effect of polymyxin B (PMB) on bovine IL-8 mRNA expression. Bovine AMs were cultured in the presence of 1 μg of P. haemolytica LPS per ml with or without preincubation of 10 μg of polymyxin B per ml. (A) After 1 h, the cells were harvested and Northern blot analysis was performed as described in Materials and Methods. (B) Relative levels of IL-8 mRNA were normalized to GAPDH. The data shown are representative of three separate experiments.

FIG. 4

Time course of bovine IL-8 mRNA expression. Bovine AMs were stimulated with 1 μg of P. haemolytica LPS per ml for the indicated times. (A) Northern blot analysis with the IL-8 probe was performed as described in Materials and Methods. (B) Relative levels of hybridization signal were normalized to GAPDH. The data shown are representative of three separate experiments. (C) The results are expressed as a percentage of the band representing the maximal intensity for each blot. The final results represent mean ± SEM (n = 3).

FIG. 5

IL-8 mRNA expression is induced by recombinant human TNF-α. (A) After 4 h, the cells were harvested and Northern blot analysis was performed as described in Materials and Methods. HIA, 20 ng of rhTNF-α per ml was boiled for 30 min before being added to AMs. (B) Relative levels of IL-8 mRNA were normalized to GAPDH. The data shown are representative of two separate experiments.

FIG. 6

Time course of bovine TNF-α mRNA expression. Bovine AMs were stimulated with 1 μg of P. haemolytica LPS per ml for the indicated times. Northern blot analysis with the bovine TNF-α probe was performed as described in Materials and Methods.

FIG. 7

Time-dependent secretion of extracellular TNF-α by bovine AMs stimulated with 1 μg of P. haemolytica LPS per ml for the indicated times, as assessed by a bioassay. The TNF-α concentration was calculated by using the mean absorbance of duplicate wells and is expressed in picograms per milliliter as described in Materials and Methods. The values shown represent mean ± SEM (n = 3).

FIG. 8

Inhibition of bovine IL-8 mRNA expression with monospecific polyclonal antibodies against bovine TNF-α. (A) Northern blot analysis with the IL-8 probe was performed as described in Materials and Methods. (B) Relative levels of IL-8 mRNA were normalized to GAPDH mRNA levels. The data shown are representative of two separate experiments. PIS, preimmune serum.