Cytokine-binding and acute-phase plasma proteins in pigs

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Singh, Harmanjit
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University of Guelph

The present studies were undertaken to look for cytokine binding plasma proteins that might be involved in resistance or susceptibility to the inflammatory responses of pigs exposed to Mycoplasma hyorhinis, Actinobacillus pleuropneumoniae or Escherichia coli lipopolysaccharide (LPS). Samples of plasma, serum or inflammatory exudates from these pigs were subjected to PAGE after incubation under covalent cross-linking conditions with human recombinant (hr) \sp125I-labelled forms of several pro-inflammatory cytokines. TGF-$\beta1,interleukin−8(IL−8\sb{72})$ and RANTES were found to bind porcine alpha2-macroglobulin (α\sb2M) in plasma and in M. hyorhinis-induced exudates. The α\sb2M in these exudates remained in the electrophoretically "slow" or native form suggesting that the proteinase-activated "fast" forms of α\sb2M are not present in these inflammatory exudates. Various members of the chemokine family of leukocyte chemoattractants were examined under the same conditions. In pigs infected with M. hyorhinis, there was a marked induction of the acute phase response, evident by elevations in plasma haptoglobin. Also induced in plasma and exudates was a major protein (120-130 kDa) that preferentially bound to chemokines of the C-C family (RANTES and MIP-1β). This protein(s) also bound moderately to IL-8, a member of C-X-C family, but not to GROα, which instead appeared to bind to a protein of approximately 8 kDa size present in all serum samples. The inducible 120-130 kDa chemokine binding protein was much more abundant in plasma of pigs from a high immune response genetic line, which underwent a more marked acute phase haptoglobin response to M. hyorhinis compared to pigs of a low immune response line. IL-1β also bound to a minor extent in the 120-130 kDa region, whereas TGF-$\beta$1 and interleukin-5 (IL-5) did not. Because the inducible chemokine binding proteins appeared coinduced with other acute phase proteins in pigs with M. hyorhinis, the spectrum of acute phase proteins of pigs was examined. After exposure of pigs to E. coli LPS, we isolated and cultured the hepatocytes in \sp35S-methionine and subjected medium to 2D-PAGE and autoradiography to detect LPS-induced secreted proteins. This approach revealed that major LPS-induced proteins of pigs were: p120 (consistent with the major acute phase protein of pigs); p90-100 (ceruloplasmin); p46 (haptoglobin); p70 (fibrinogen); p75-77 ($\alpha$1-antichymotrypsin) and a number of unidentified proteins in the 25-30 kDa range. Notable downregulated proteins were p79 (transferrin); p48-50 (consistent with $\alpha$1-acid glycoprotein) and p70 (consistent with $\alpha$1-antitrypsin). Collectively, these studies indicate that pigs have several acute phase proteins, including an inducible chemokine binding protein that might be involved in regulating leukocyte responses in bacterial infections. Since this chemokine binding protein is preferentially expressed in pigs selected for stronger immune responsiveness, the chemokine binding protein might be a marker for reduced susceptibility to phagocyte dependent tissue damage in bacterial infections.

pigs, cytokine-binding plasma proteins, acute-phase plasma proteins, Mycoplasma hyorhinis, inflammatory responses