© 2005 American Heart Association, Inc.
Letters to the Editor |
Letter to the Editor
In response to van den Berg et al:
Department of Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands
We appreciate the interest of van den Berg and Taylor1 in our article on the direct actions of CRP in humans.2 In the context of numerous in vitro studies demonstrating that CRP elicits proatherogenic changes, our study was designed to examine the bioactivities of CRP in a human setting.
Van den Berg states that filtration methods are not very rigorous in removing endotoxin. However, the currently used purification process was successful in lowering the endotoxin burden by >97.5% within the recombinant human CRP (rhCRP) preparation to levels insufficient to cause any bioactivity in vivo, as demonstrated by standard methods. The final purified product was >97% pure by high-pressure size-exclusion chromatography (SEC-HPLC) and reverse-phase HPLC, whereas Time-of-Flight mass spectrometry provided supporting data for high purity, showing no other protein fractions besides the rhCRP.
Second, van den Berg poses that the kinetics of cytokine production observed on CRP-infusion fit the cytokine profile of endotoxin contamination. However, on comparison to endotoxin activities in humans, we show a clear disparity in cytokine profiles between that mediated by CRP and endotoxin.2,3 Administration of endotoxin to animals as well as humans is invariably associated with rapid induction of TNF
, a central and obligatory mediator that orchestrates a consistent sequence of events regarding cytokine production and coagulation activation.3,4 Markedly, throughout all human CRP protocols, we were unable to detect any change in TNF expression, either at leukocyte mRNA levels or circulating plasma levels. Furthermore, heat-inactivation of the CRP preparation significantly diminished the capacity of CRP to evoke an inflammatory response in whole blood stimulation assays (Bisoendial, data on file). This finding, which is consistent with data from other investigators,5,6 demonstrates again that CRP itself is largely responsible for the proinflammatory effects rather than potential contaminants in this highly purified rhCRP preparation.
Finally, van den Berg questions the kinetics of CRP levels, which appears to be inconsistent with levels of IL-6 and IL-8 which decrease from 4 hours onwards. Apart from the disparity in half-life between CRP and measured cytokines, several aspects of the inflammatory response merit further clarification. Thus, CRP induces the secretion of cytokines from arterial endothelium and monocyte macrophages.5,6 Cumulated evidence suggests that these effects are receptor-mediated. Deveraj et al recently demonstrated that CRP exerts its biological activities in human aortic endothelial cells (HAEC) via binding and internalisation through Fc
receptors, CD32 and CD64.7 Similarly, biological activity on ligand-receptor engagement has been shown by other investigators in monocytes.8 In this regard, saturable binding and/or uptake of CRP in HAECs as well as leukocytes may have blocked further cytokine production in our study. An alternative explanation for the temporary cytokine response despite persistently high CRP levels involves activation of potent counter-regulatory systems, including IL-10 and induction of A20 (data on file). Moreover, CRP has been shown to regulate soluble IL-6 receptor (sIL-6R) shedding in human neutrophils and markedly increase sIL-6R/IL-6 complex formation.9 Apart from the biological responses that IL-6 may elicit via its receptor, the IL-6/sIL-6R complex may perpetuate its own biological activity by binding the signal-transducing component of the IL-6R complex. Infusion of rhIL-6 was recently shown to be associated with a delayed increase in plasma CRP in humans, which was sustained for more than 16 hours.10 Thus, self-perpetuating mechanisms in view of inflammatory responses are not uncommon, and the IL-6 increase may conceivably have resulted in the second rise in CRP levels after 24 hours.
In conclusion, we concur with van den Berg that caution is mandatory with respect to the use of purified commercial rhCRP in experimental studies. However, the currently available data provide compelling evidence that CRP itself mediates proinflammatory and prothrombotic effects in vivo in humans. Within the next few years, novel strategies, directed at specifically targeting CRP-activity, probably may enable us to further elucidate the biological relevance of these actions in humans.
References
1. van den Berg CW, Taylor KE. Activation of inflammation and coagulation after infusion of C-reactive protein in humans. Circ Res. 2005; 97: e2.
2. Bisoendial RJ, Kastelein JJ, Levels JH, Zwaginga JJ, van den BB, Reitsma PH, Meijers JC, Hartman D, Levi M, Stroes ES. Activation of inflammation and coagulation after infusion of C-reactive protein in humans. Circ Res. 2005; 96: 714–716.
3. van Deventer SJ, Buller HR, ten Cate JW, Aarden LA, Hack CE, Sturk A. Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood. 1990; 76: 2520–2526.
4. Haudek SB, Natmessnig BE, Furst W, Bahrami S, Schlag G, Redl H. Lipopolysaccharide dose response in baboons. Shock. 2003; 20: 431–436.[CrossRef][Medline] [Order article via Infotrieve]
5. Devaraj S, Kumaresan PR, Jialal I. Effect of C-reactive protein on chemokine expression in human aortic endothelial cells. J Mol Cell Cardiol. 2004; 36: 405–410.[CrossRef][Medline] [Order article via Infotrieve]
6. Ballou SP, Lozanski G. Induction of inflammatory cytokine release from cultured human monocytes by C-reactive protein. Cytokine. 1992; 4: 361–368.[CrossRef][Medline] [Order article via Infotrieve]
7. Devaraj S, Du Clos TW, Jialal I. Binding and internalization of C-reactive protein by Fcgamma receptors on human aortic endothelial cells mediates biological effects. Arterioscler Thromb Vasc Biol. 2005; 25: 1359–1363.
8. Woollard KJ, Phillips DC, Griffiths HR. Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells. Clin Exp Immunol. 2002; 130: 256–262.[CrossRef][Medline] [Order article via Infotrieve]
9. Jones SA, Novick D, Horiuchi S, Yamamoto N, Szalai AJ, Fuller GM. C-reactive protein: a physiological activator of interleukin 6 receptor shedding. J Exp Med. 1999; 189: 599–604.
10. Steensberg A, Fischer CP, Keller C, Moller K, Pedersen BK. IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans. Am J Physiol Endocrinol Metab. 2003; 285: E433–E437.
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