A comparison of equine and bovine sera as sources of lipopolysaccharide-binding protein activity in equine monocytes incubated with lipopolysaccharide.
Abstract: Lipopolysaccharide-binding protein (LBP) is an acute phase protein that binds the lipid A moiety of lipopolysaccharide (LPS) and transfers LPS monomers to soluble CD14 in plasma or membrane bound CD14 on mononuclear phagocytes. The result of these interactions is activation of the TLR4 receptor complex, and the synthesis and release of inflammatory mediators. Inclusion of LBP in cellular assays increases the sensitivity of cells expressing CD14 to LPS. Therefore, the objectives of this study were to (1) compare differentially treated sera from cattle and horses as sources of LBP activity using LPS-induced expression of procoagulant activity (PCA) by equine monocytes as a readout and (2) evaluate the use of commercial equine serum as a source of LBP activity using LPS concentration response and time course studies to validate the response. Monocytes were isolated from eight horses and incubated with five different serum preparations in the presence or absence of Escherichia coli LPS. The sera tested were heat-inactivated fetal bovine serum (HI-FBS), pooled commercial equine serum (CES), heat-inactivated pooled commercial equine serum (HI-CES), autologous equine serum (AES), and heat-inactivated autologous equine serum (HI-AES). In the absence of LPS, monocytes from half of the horses in the study had increased expression of PCA when incubated with HI-FBS alone; PCA was unaffected by incubation with the other sera. There was a four-fold increase in PCA when monocytes were incubated with LPS in the presence of CES, HI-CES or AES compared to LPS without serum. The combination of HI-FBS and LPS increased PCA 20-fold compared to LPS without serum. The HI-AES serum lacked significant LBP activity. Whereas maximal expression of PCA was induced by 1ng/ml of LPS in the absence of serum, inclusion of 1% CES reduced the LPS concentration required for maximal PCA to 30pg/ml. Monocytes incubated with LPS in the presence of CES had increased PCA at 3h and peaked at 6h. In conclusion, monocytes from many horses are directly stimulated by HI-FBS, suggesting that HI-FBS is not an optimal source of LBP for in vitro studies of LPS with equine monocytes. In contrast, CES and AES are effective sources of LBP activity for such studies, as they do not directly induce activation. Although the heat inactivation process did not affect the LBP activity in CES, it ablated LBP activity in AES. Consequently, investigators are advised to utilize either CES or AES in future studies, but not heat-inactivated AES.
Publication Date: 2007-10-10 PubMed ID: 18023485DOI: 10.1016/j.vetimm.2007.10.002Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article analyses the abilities of different sera from horses and cattle to activate an immune response component called Lipopolysaccharide-binding protein (LBP) in horse monocytes. The study concludes that non-heat treated commercial and autologous equine sera are more effective sources of LBP, whereas heat-treated fetal bovine serum and autologous equine serum are less effective.
Role and Significance of Lipopolysaccharide-binding protein (LBP)
- LBP is a protein produced during inflammatory responses which binds with lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria such as Escherichia coli.
- LBP transfers LPS monomers to a receptor known as CD14, either soluble in plasma or bound to the membrane of mononuclear phagocytes (type of white blood cells).
- This interaction activates the TLR4 receptor complex, leading to the production and release of inflammatory mediators.
- In short, LBP plays a crucial role in recognising and responding to bacterial infections. In this context, the ability of different sera to enhance LBP activity was of interest to the researchers.
Experiment Parameters and Methodology
- Different types of serum were tested for their ability to activate the expression of Procoagulant Activity (PCA) by horse monocytes, a sign of an immune response.
- These sera were from cattle (specifically, Heat-Inactivated Fetal Bovine Serum, or HI-FBS) and horses (Autologous Equine Serum, Commercial Equine Serum, Heat-Inactivated versions of both).
- Monocytes were isolated from eight horses and combined with these different sera in the presence or absence of Escherichia coli LPS as a stimulant.
Results and Conclusions
- Monocytes incubated with heat-inactivated fetal bovine serum showed increased PCA expression even without LPS stimulus, suggesting that it can directly stimulate monocytes. This makes it less suitable for LBP studies as it can give misleading results.
- When mixed with LPS, monocytes showed four-fold PCA increase in the presence of equine serum (both commercial and autologous) compared to LPS alone.
- However, findings showed a whopping 20-fold increase in PCA when mixed with heat-inactivated fetal bovine serum and LPS.
- Surprisingly, heat-inactivated autologous equine serum showed little LBP activity, suggesting that the heat-inactivation process can affect LBP activity depending on the source.
- The study concluded that non-heat treated commercial and autologous equine sera are more effective sources of LBP. Heat-treated autologous equine serum should be avoided in future studies due to its reduced LBP activity.
Cite This Article
APA
Figueiredo MD, Salter CE, Hurley DJ, Moore JN.
(2007).
A comparison of equine and bovine sera as sources of lipopolysaccharide-binding protein activity in equine monocytes incubated with lipopolysaccharide.
Vet Immunol Immunopathol, 121(3-4), 275-280.
https://doi.org/10.1016/j.vetimm.2007.10.002 Publication
Researcher Affiliations
- Department of Large Animal Medicine, The University of Georgia, Athens, GA 30602-7385, USA. mf239@uga.edu
MeSH Terms
- Acute-Phase Proteins / immunology
- Animals
- Blood Coagulation Factors / immunology
- Carrier Proteins / blood
- Carrier Proteins / immunology
- Cattle / blood
- Cattle / immunology
- Horses / blood
- Horses / immunology
- Linear Models
- Lipopolysaccharide Receptors / immunology
- Lipopolysaccharides / immunology
- Lipopolysaccharides / pharmacology
- Membrane Glycoproteins / blood
- Membrane Glycoproteins / immunology
- Monocytes / immunology
Citations
This article has been cited 2 times.- Naskou MC, Sumner SM, Chocallo A, Kemelmakher H, Thoresen M, Copland I, Galipeau J, Peroni JF. Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells. Stem Cell Res Ther 2018 Mar 22;9(1):75.
- Schmidt D, Joyce EJ, Kao WJ. Fetal bovine serum xenoproteins modulate human monocyte adhesion and protein release on biomaterials in vitro. Acta Biomater 2011 Feb;7(2):515-25.
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