Elsevier

Immunology Letters

Volume 96, Issue 2, 31 January 2005, Pages 203-210
Immunology Letters

Epidermal growth factor enhances TNF-α-induced priming of human neutrophils

https://doi.org/10.1016/j.imlet.2004.08.012Get rights and content

Abstract

The intensity of neutrophil inflammatory response could be rapidly amplified by priming with pro-inflammatory mediators such as TNF-α, GM-CSF or LPS at low concentrations prior to stimuli. We proposed that epidermal growth factor (EGF) increases TNF-α-induced priming of human neutrophils. This study showed that EGF enhanced TNF-α-induced activation of neutrophils functions. The addition of EGF to neutrophils cultured with TNF-α resulted in increased respiratory burst and phagocytic activity of polymorphonuclear leukocytes (PMN) and up-regulation of adhesion molecule CD11b. Moreover, EGF enhanced IL-8 production by TNF-α-primed PMN. EGF alone was able to prime CD11b expression and IL-8 production by PMN. EGF receptor selective tyrosine kinase inhibitor, tyrphostin AG-1517, blocked the effect of priming with EGF, whereas the status of non-primed and TNF-α-primed neutrophils remained unaffected. EGFR expression on neutrophils was confirmed by flow cytometry and CELISA methods. These data provide the original evidence that EGF significantly enhances TNF-α-induced priming of human neutrophils acting through EGFR tyrosine kinase pathway. The observed effect may be a result of co-operative action of EGF, TNF-α and reactive oxygen intermediates (ROI).

Introduction

Neutrophils constitute a fundamental component of the innate immune response. Peripheral blood polymorphonuclear leukocytes (PMN) could be primed for increased response by pro-inflammatory mediators such as TNF-α, IL-8, GM-CSF, C5a, leukotrien B4 (LTB4), INF-γ, LPS, that prepare neutrophils for enhanced response to following stimulation (1). Priming is crucial for optimising neutrophil response and enhancing the killing of pathogens. The principle consequence of priming, aside from a direct effect on cell polarization, deformability and integrin/selectin expression, is the readiness of cells for increased reactive oxygen intermediate (ROI) generation and degranulation of granular proinflammatory mediators in response to the subsequent stimuli [1], [2]. The mechanisms of PMN priming are not fully understood. Redistribution of the NADPH oxidase components may be the basis of the LPS and TNF-α-induced priming, and p38 mitogen-activated protein kinase (MAPK) has been shown to play a role in regulation of flavocytochrome b558 expression [3], [4]. Partial phosphorylation of the cytosolic oxidase component p47phox has been demonstrated during LPS and GM-CSF-induced priming [3], [5]. Activation of MAPK cascades is implicated in priming trigged by TNF-α, LPS and GM-CSF [6], [7], [8], [9], [10], [11], [12], [13], and it is correlated with tyrosine phosphorylation [12], [14], [15], [16], [17].

Epidermal growth factor (EGF) and EGF-like protein family play an important role both in physiological processes like embriogenesis, cell proliferation and differentiation, wound healing, as well as in pathological conditions such as atherosclerosis or neoplasmic diseases [18], [19], [20], [21]. EGF is produced in blood vessels mainly by platelets and endothelial cells. The presence of EGF together with inflammatory and/or growth factors raises the possibility that EGF may influence the function of inflammatory cells. The EGF receptor (EGFR) is a member of the tyrosine kinase receptor family, which is expressed on a variety of cells and plays an important role in cell division and tumor growth [22], [23]. The EGF receptor is composed of an extracellular domain with a binding capacity, a transmembrane domain and an intracellular domain with tyrosine kinase activity. Binding of the agonist to the receptor triggers a cascade of events that includes receptor autophosphorylation, phosphorylation of intracellular substrates and activation of signaling pathways. Several reports have demonstrated that stimuli, unrelated to EGF-like ligands, such as TNF-α or ROI, can also activate the EGF receptor [24], [25], [26], [27], [28], [29]. It has been described that EGF enhances TNF-α-induced NF-κB activation in NIH3T3 cells in a dose-dependent manner [25]. TNF-α has a costimulatory effect on heparin-binding epidermal growth factor (HB-EGF), EGF, TGF-α and β-cellulin-induced DNA synthesis in stromal cells [26]. Together, this evidence points to the possibility of cross-talk between TNF-α and EGF signalling pathways in primed neutrophils. The role of the growth factors in the priming of PMN has not been explored sufficiently until now. Pillinger et al. have previously demonstrated that EGF has no effect on neutrophil p42/p44 MAPK activation, suggesting the lack of EGFR on PMN [30]. Similarly, Vinante et al. have shown the lack of EGFR on neutrophils using flow cytometry analysis [31]. We hypothesize, however, that EGF influences the neutrophil function by amplifying signalling pathways trigged by TNF-α.

In this study we analysed the effect of EGF on PMN function. We have shown that EGF enhances TNF-α-induced priming of neutrophils in vitro, and selective EGFR tyrosine kinase inhibitor tyrphostin AG-1517 attenuate EGF-mediated signalling. These data provide evidence for EGF involvement in PMN activation which is dependent on EGFR tyrosine kinase activation.

Section snippets

Isolation of neutrophils

Human neutrophils were isolated from heparinized peripheral blood obtained from healthy volunteers who had abstained from taking any drugs for at last 3 weeks. Neutrophil isolation was performed using a one-step density-gradient centrifugation on Polymorphprep (Nycomed Pharma AS, Oslo, Norway). After two washings with PBS, neutrophils were maintained in PBS or in RPMI 1640 supplemented with 10% FCS (Sigma–Aldrich, Saint Louis, USA) and 1% penicillin and streptomycin (Sigma–Aldrich). After the

Neutrophils express EGF receptor

To determine whether the EGFR are present on neutrophils surface, we used flow cytometry and cellular enzyme-linked immunosorbent assay. Despite previous suggestion that myeloid cells do not express EGFR, we detected EGFR on neutrophils using two independent methods (Fig. 1). EGFR expression increased significantly after incubation of neutrophils with TNF-α, EGF + TNF-α, and LPS.

EGF enhances ROI production by TNF-α-primed PMN

To examine the effect of EGF on respiratory burst of PMN, cells were primed with TNF-α and EGF in different

Discussion

Activation of neutrophils depends on the proinflammatory mediators released in the inflammatory site, which intensify neutrophil response. TNF-α is one of the best explored mediator of PMN activation in vitro. At low concentrations (1–100 ng/ml), TNF-α has no visible effect on neutrophils, but subsequent addition of another stimulator to PMN suspension contributes to much higher activation of PMN effector functions (1). We hypothesis that some growth factors released in the initial period of

Acknowledgment

This work was supported by a grant from the State Committee for Scientific Research of Poland: KBN 3 P05A 06725.

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