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 <i>in vitro</i> 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 <math> <f> <g>m</g></f> </math>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 <i> in vivo</i> 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.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10212004-001455 |
| Date | 01 January 1999 |
| Creators | Caswell, Jeffrey Llewellyn |
| Contributors | Middleton, Dorothy M., Gordon, John R. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-10212004-001455 |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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