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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 18, 2003 as doi:10.1096/fj.02-1000fje. |
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Centre for Cardiovascular Biology and Medicine, School of Biomedical Sciences, King’s College, New Hunt’s House, Guy’s Campus, London, UK;
* Clinical Pharmacology, Barts and the London, Queen Mary’s School of Medicine, London, UK
2Correspondence: Centre for Cardiovascular Biology and Medicine, King’s College, New Hunt’s House, Guy’s Campus, London, SE1 1UL, UK. E-mail: sue.brain{at}kcl.ac.uk
SPECIFIC AIMS
The aim of this study was to investigate the mechanisms via which extravasated neutrophils become involved in mediating the thermal hyperalgesia induced by nerve growth factor (NGF), which failed to directly activate neutrophils but by comparison, stimulated endothelial cells with resulting up-regulation of intercellular adhesion molecule-1 (ICAM-1). This in turn acted in an essential manner to enable neutrophil accumulation and as a consequence, inflammatory hyperalgesia. Thus, a central role of the postcapillary venule endothelial cell in initiating thermal hyperalgesia is revealed in vivo, which links the vascular and neural inflammatory components.
PRINCIPAL FINDINGS
1. NGF induces neutrophil accumulation in the rat and mouse via a neutrophil-dependent mechanism that is independent of the sensory neuropeptide neurokinin 1 (NK1) receptor
Initial experiments confirmed previous findings that NGF-induced thermal hyperalgesia in the rat is dependent on circulating neutrophils, as determined by a loss of response in rats depleted of circulating neutrophils with a rat antineutrophil antiserum. NGF is established as a sensory growth factor. The best-established, proinflammatory sensory neuropeptide is substance P and its receptor, the NK1. We investigated the involvement of NGF-induced responses in wild-type and tachykinin NK1 receptor knockout mice, which lack some algesic responses and the ability to mediate the plasma extravasation and neutrophil accumulation associated with neurogenic inflammation. However, the results show that the neutrophil accumulation in addition to the thermal hyperalgesia induced by NGF is similar in wild-type and NK1 knockout mice and thus, is independent of the involvement of the sensory neuropeptide NK1 receptor.
2. NGF does not activate isolated neutrophils in vitro
Arachidonate 5-lipoxgenase inhibitors have previously been shown to block NGF-induced, inflammatory hyperalgesia. The present study provides evidence that the 5-lipoxygenase products leukotriene B4 (LTB4) and 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid, like NGF, are able to mediate thermal hyperalgesia in the rat paw. This led us to hypothesize that a LT released from activated neutrophils is the final mediator of NGF-induced hyperalgesia. It is well established that neutrophils release 5-lipoxygenase products, and thus, we investigated the ability of NGF to activate neutrophils, which, elicited from the rat peritoneal cavity, were incubated in vitro for 30 min or 4 h with calcium ionophore or NGF. Calcium ionophore released significant amounts of immunoreactive LTB4 and myeloperoxidase (MPO). Thromboxane B2 levels were also measured for use as an alternative marker of eicosanoid production from activated neutrophils. NGF was not able to stimulate release of any agent. The lack of ability of NGF to stimulate eicosanoid or MPO release in vitro led us to conclude that NGF cannot directly act to stimulate neutrophils.
3. NGF stimulates neutrophil accumulation and thermal hyperalgesia in vivo by mechanisms dependent on de novo protein synthesis and up-regulation of the endothelial cell adhesion molecule ICAM-1
NGF induces neutrophil accumulation in a time-dependent manner over 0–5 h, in keeping with established time courses of neutrophil accumulation, which are observed secondary to the up-regulation of endothelial cell adhesion molecules via a de novo protein synthesis-dependent mechanism. The possibility that neutrophil accumulation occurred secondary to endothelial cell activation was investigated. The neutrophil accumulation and thermal hyperalgesia induced by NGF were blocked by actinomycin D, a mRNA transcription inhibitor, suggesting that NGF-induced protein biosynthesis is necessary for the response. The participation of endothelial cells in the microvascular response to NGF was confirmed by experiments involving an antibody to the major endothelial cell-derived ICAM-1. The results shown in Figure 1
demonstrate that pretreatment [intravenously (i.v.)] of anti-ICAM-1 monoclonal antibody (mAb; 2 mg/kg) 15 min before administration of NGF significantly reduced the neutrophil accumulation (see Fig. 1a
), and pretreatment with an irrelevant antibody (2 mg/kg) or vehicle control did not compromise the neutrophil accumulation. A similar pretreatment with anti-ICAM-1 mAb abolished NGF-induced thermal hyperalgesia (Fig. 1b
) with NGF-inducing hyperalgesia in the presence of irrelevant antibody. Our finding that NGF stimulates the up-regulation of ICAM-1 in human cultured vascular endothelial cells in the absence of neutrophils supports these results.
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CONCLUSIONS AND SIGNIFICANCE
NGF levels have been shown to be increased systemically or locally in inflamed tissue in a range of diseases that include rheumatoid arthritis, skin diseases, and asthma. The established relationship of NGF with sensory nerves has prompted the suggestion that NGF mediates its potent hyperalgesic effects via sensory nerves or the neuropeptides that they release. The present results provide little support for the hypothesis that the major substance P NK1 receptor is involved in the neutrophil-dependent hyperalgesia, as responses were similar in wild-type and NK1 knockout mice, and support previous findings using NK1 receptor antagonists.
Thus, we hypothesized that NGF acted independently of substance P to stimulate neutrophil accumulation and that the neutrophils then emigrate into skin, where they are activated to release LTB4. However, our results show that NGF failed to activate rat-isolated neutrophils in vitro. This was surprising, as NGF, extracted from mouse salivary glands, has been shown to be chemotactic for neutrophils in vivo and in vitro, acting via a complement-independent pathway. Our conflicting results may be related to the fact that the murine 7S recombinant NGF used in this study may not possess some of the factors that NGF purified from saliva, used in previous studies, contains. Alternatively, our results may be related to a lack of functional NGF receptor TrkA on polymorphonuclear leukocytes. Thus, there is far-from-conclusive evidence to suggest that neutrophils possess a functional NGF receptor necessary to activate neutrophils. The source of the 5-lipoxygenase products remains unclear, but they may well have a direct effect on sensory nerves that mediate the hyperalgesia, in that it is shown that they can act as agonists at the vanilloid receptor subtype 1 on sensory neurons in culture.
The use of protein synthetase/mRNA inhibitor actinomycin D and an antibody to ICAM-1 provided evidence that NGF stimulates de novo protein, synthesis-dependent up-regulation of endothelial adhesion molecule expression, which in turn, leads to neutrophil accumulation and emigration into extravascular tissues. The effect of NGF on neutrophil accumulation and hyperalgesia was shown to be blocked by pretreatment with actinomycin D and a mAb to ICAM-1. The results demonstrating that NGF up-regulates ICAM-1 in rat skin microvasculature and on human umbilical vein endothelial cells are complimentary to the demonstration by others of the high-affinity TrkA receptor, as well as the low-affinity p75 receptor on endothelial cells. Our results provide novel evidence that a major, functional consequence of the ability of NGF to activate endothelial cells in vivo is to mediate neutrophil accumulation and as a consequence, thermal hyperalgesia (Fig. 2
).
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The results demonstrate that the neutrophils accumulate in response to NGF via an endothelial-dependent step involving the major adhesion molecule ICAM-1. The functional extent of the consequences of the ability of picomolar amounts of NGF to stimulate endothelial cell adhesion molecule and neutrophil accumulation is at present unclear. We have recently shown that macrophage and lymphocyte numbers, in addition to hyperalgesia, increase at NGF-pretreated sites in a neutrophil-dependent manner. The evidence supports the concept that therapeutic interventions that block inappropriate NGF generation may play vital roles in the treatment of inflammatory disease and the associated pain. In conclusion, these results identify a central role of the postcapillary venular endothelial cell in playing an essential role in initiating thermal hyperalgesia induced by NGF in the rat.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1000fje; doi: 10.1096/fj.02-1000fje 
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), irrelevant antibody (
), or anti-ICAM-1 pretreatment (
). Results are taken as paw withdrawal latency [P.W.L. (s)], compared with the contralateral uninjected paw with significance, compared with preinjected values. Results are shown as mean ± SE, n = 6, with significance (**, P<0.01), compared with preinjected sites. i.pl., Intraplantar.