Abstract
The serum amyloid A (SAA) gene family is highly conserved and encodes acute phase proteins that are upregulated in response to inflammatory triggers. Over the years, a considerable amount of literature has been published attributing a wide range of biological effects to SAAs such as leukocyte recruitment, cytokine and chemokine expression and induction of matrix metalloproteinases. Furthermore, SAAs have also been linked to protumorigenic, proatherogenic and anti-inflammatory effects. Here, we investigated the biological effects conveyed by murine SAA3 (mu rSAA3) recombinantly expressed in Escherichia coli. We observed the upregulation of a number of chemokines including CCL2, CCL3, CXCL1, CXCL2, CXCL6 or CXCL8 following stimulation of monocytic, fibroblastoid and peritoneal cells with mu rSAA3. Furthermore, this SAA variant displayed potent in vivo recruitment of neutrophils through the activation of TLR4. However, a major problem associated with proteins derived from recombinant expression in bacteria is potential contamination with various bacterial products, such as lipopolysaccharide, lipoproteins and formylated peptides. This is of particular relevance in the case of SAA as there currently exists a discrepancy in biological activity between SAA derived from recombinant expression and that of an endogenous source, i.e. inflammatory plasma. Therefore, we subjected commercial recombinant mu rSAA3 to purification to homogeneity via reversed-phase high-performance liquid chromatography (RP-HPLC) and re-assessed its biological potential. RP-HPLC-purified mu rSAA3 did not induce chemokines and lacked in vivo neutrophil chemotactic activity, but retained the capacity to synergize with CXCL8 in the activation of neutrophils. In conclusion, experimental results obtained when using proteins recombinantly expressed in bacteria should always be interpreted with care.
Highlights
Contamination of medical drugs with bacterial products has been a constant concern long before recombinant technology was introduced
Lewis lung carcinoma (LLC) cells were stimulated for a period of 16 h with different concentrations of the inflammatory mediators LPS, IL-1β or TNFα, whereafter SAA3, SAA1 and CCL2 mRNA were determined by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) (Figures 1(a)–1(c))
SAA1 mRNA was highly induced by LPS, whereas this transcript was upregulated to a smaller extent when LLC cells were treated with IL-1β, reaching a maximal upregulation of the SAA1 gene of 25 ± 9-fold at 10 ng/ml of IL-1β (n = 3; p < 0:05) (Figure 1(b))
Summary
Contamination of medical drugs with bacterial products has been a constant concern long before recombinant technology was introduced. Lipopolysaccharide (LPS) from Gramnegative bacteria such as Escherichia coli (E. coli) is of particular relevance. Such endotoxins provoke fever and other inflammatory effects upon systemic or local injection. Interferon (IFN) produced in vitro in fibroblasts or leukocytes was found to be pyrogenic upon injection and this was long thought to be mediated by traces of LPS. Following purification to homogeneity, IFN caused fever and could be considered as an endogenous pyrogen [2]. The fact that IFN was introduced for medical treatment forced the pharmaceutical industry to deal with LPS contamination [6, 7]
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