The role of interleukin-8 as a neutrophil chemoattractant In bovine pneumonic pasteurellosis
Caswell, Jeffrey Llewellyn
The role of interleukin-8 (IL-8) as a neutrophil chemoattractant in bovine pneumonic pasteurellosis was investigated by characterizing: (1) the expression of IL-8 in the lesions of pneumonic pasteurellosis, (2) the ' in vitro' and 'in vivo' effects of recombinant bovine IL-8 on neutrophil chemotaxis, and (3) the importance of IL-8 as a neutrophil chemoattractant in this disease. The expression of IL-8 in bovine pneumonic pasteurellosis was assessed by Northern analysis, 'in situ' hybridization and enzyme-linked immunosorbent assay. The expression of IL-8 mRNA was elevated dramatically in lesional lung compared to non-lesional lung, viral pneumonia, and normal lung. 'In situ' hybridization revealed intense IL-8 mRNA expression in alveolar macrophages and neutrophils, and milder expression in several other cell types. Bronchoalveolar lavage (BAL) fluid from lesional lung contained 16.06 ± 4.00 ng/ml IL-8, but lower levels were present in non-lesional lung, viral pneumonia, and normal lung (mean ± SEM). Recombinant bovine IL-8 (rbIL-8) was produced using a pGEX expression system. The rbIL-8 induced in vitro chemotaxis and shape change of bovine neutrophils at doses as low as 6.3 ng/ml. After injection of 1.0 ng to 3.3 m g of rbIL-8 into bovine skin, neutrophils marginated in vessels within 15 minutes and infiltrated the dermis by 1 hour after injection. These results demonstrate that bovine IL-8 is a neutrophil chemoattractant 'in vitro ' and 'in vivo', at levels equivalent to those present in pneumonic pasteurellosis. An ovine model of pneumonic pasteurellosis was developed to ascertain the effects of in vivo neutralization of IL-8 on the development of disease. At 0, 3, 8 and 20 hours after bacterial infection (p.i.), BAL fluid samples contained 6.5 ± 2.4, 110 ± 48, 554 ± 118, and 507 ± 70 ng/ml IL-8, respectively. Neutrophil infiltration was mild at 3 hours p.i. but progressively more intense at 8 and 20 hours p.i. Lambs were treated intravenously with up to 80 mg of an IL-8-neutralizing antibody prior to bacterial challenge. The antibody treatment failed to completely neutralize IL-8 activity, probably because the levels of IL-8 in the lung were unexpectedly high. The contribution of IL-8 to the neutrophil chemotactic activity of BAL fluid from cattle and lambs with pneumonic pasteurellosis was assessed. Pre-treatment of pneumonic BAL fluid samples with a neutralizing antibody to IL-8, compared to pretreatment with a control antibody, reduced the 'in vitro' neutrophil chemotactic activity significantly, by 15-63% in 9/10 samples. An extract of pneumonic bovine lung induced vigorous neutrophil infiltration following injection into bovine skin, and depletion of IL-8 from the extract reduced this neutrophil influx by 60%. These data indicate that IL-8 is an important neutrophil chemoattractant in both early and well-developed lesions of pneumonic pasteurellosis, but that mediators with actions redundant to those of IL-8 must also be present at these times.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeMiddleton, Dorothy M.; Gordon, John R.
Copyright DateSeptember 1998