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- PMID: 19388520
Abstract
The aim of this study was to evaluate the effects of lactoferrin (LF) on the growth of fibroblasts derived from nasal polyps. We showed that the proliferation of fibroblasts was inhibited in a dose-dependent manner by both native and recombinant LF. The greatest inhibition of proliferation was caused by human milk-derived, iron-saturated LF. The inhibition of fibroblast proliferation was not species specific because bovine LF also was active. The interaction between LFs and a putative cell receptor did not depend on the sugar composition of the glycan moiety of the LF molecule because lactoferrins of different origins were active and the addition of monosaccharides to the cultures did not block proliferation. However, the treatment of fibroblasts with sodium chlorate (an inhibitor of glycosaminoglycan sulfation) or the addition of heparin abolished the inhibitory effect of LF, suggesting that LF binds heparan sulfate-containing proteoglycans. The significance of LF in nasal excretions in controlling polyp formation is discussed.
Introduction
Nasal polyposis is an inflammatory disorder of the sinonasal mucosa of unknown etiology. Chronic inflammation causes a reactive hyperplasia of the intranasal mucosal membrane, which results in the formation of polyps. Nasal polyps (NP) consist of columnar ciliated and stratified squamous epithelium covering swollen connective tissue built with fibroblasts, dilated capillaries and venous channels [29]. Upon stimulation, fibroblasts, via production of collagens and fibronectin, promote extracellular matrix generation, tissue remodeling and, consequently, stromal and epithelial abnormalities [35]. NPs are characterized by intense tissue eosinophilia that is subsequently associated with a T-helper type 2 cytokine profile [3].
Only a small proportion of NPs can be connected with genetic disorders. In the majority of cases, the etiopathology is multifactorial, often related to both the host and the environment. The factors influencing NP development include altered local homeostasis of nasal mucosa, bacterial and fungal colonization, allergy, neurovascular factors and poorly understood disturbances of the innate defense system [5].
Mucosal secretions play a crucial role in nonspecific defense against pathogens [28]. Lactoferrin (LF) represents one of the major components of mammalian secretions and plays numerous roles in innate and adaptive immunity [46]. LF belongs to the family of proteins involved in iron metabolism. The protein exists in two separate reservoirs, in body secretions [24] and in neutrophils [15]. Receptors for LF can be found in nearly all organs and cell types [38]. LF can interact with, among others, major cell receptors associated with innate immunity such as the mannose receptor [45], CD14 [13], toll-like receptors [11], CD169 sialoadhesin [10] and heparan sulfate [25].
The predominant role of LF is associated with nonspecific defense against pathogens including bacteria [8], fungi [40], viruses [36] and parasites [20]. LF regulates myelopoiesis [1], promotes maturation of T [47] and B cells [48] and exhibits adjuvant activity [17, 45]. Its important roles in host defense include antiinflammatory properties [4], in particular anti-oxidant actions [19]. LF was also found to inhibit mitogen-induced proliferation and the mixed lymphocyte reaction of human peripheral blood mononuclear cells [49], to induce apoptosis of the Jurkat leukemia T cell line [22] and to arrest the growth of human carcinoma cells [12].
LF levels are increased in nasal secretions of subjects with allergen-induced rhinitis [32]. The cellular source of both lactoferrin and lysozyme has been identified as serous cells of the submucosal glands [33]. Reports on the expression and presence of the protein in NP tissue and nasal secretions are rare and have not consistently demonstrated an increase [23, 43] or decrease [30, 31] in the LF level in polyp tissues. Additionally, no expression or downregulation of LF genes has been observed in nasopharyngeal carcinoma [42, 44]. In addition, despite higher LF expression in the submucosal tissue of NPs, the epithelial cells stained negatively for LF [43]. Hence, we assumed that the majority of reports demonstrated decreased LF levels in nasal secretions.
In view of these findings, we hypothesized that decreased expression of LF could be one of the etiological factors disturbing the local homeostasis of the nasal mucosa. Therefore, the aim of this study was to evaluate the effect of LF on the growth of NP fibroblasts and to characterize putative cell receptors for LF on cultured fibroblasts.
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Reagents
All reagents were purchased from Sigma-Aldrich unless otherwise stated. TLR2 and TLR4 antibodies were from eBioscience, pyrogen-free heparin was obtained from SERVA, and bovine milk lactoferrin (BLF) was purchased from Morinaga Milk (Tokyo, Japan). Human milk lactoferrin (HLF) and iron-saturated human milk lactoferrin (HLF-iron) were from Sigma-Aldrich (cat. No. L0520 and L3770, respectively).
NP specimens
All subjects met the diagnostic criteria for chronic rhinosinusitis as established by the Task Force on
Growth inhibition of polyp-derived fibroblasts by various molecular forms of LF
Fibroblasts isolated from NPs obtained from patients with chronic rhinosinusitis were incubated for 6 days with various molecular forms of human lactoferrin (native and iron-saturated forms) and bovine (xenogeneic) LF in the concentration range of 1–25 μg/ml (Fig. 1). Dexamethasone (DEX) (50–0.05 μg/ml) served as a reference drug. Only data on the proliferation of NP fibroblasts isolated from three patients (two males and one female), randomly chosen from a group of six patients with chronic
Discussion
In this work, we demonstrated for the first time that LF, an important element of nasal exudates, can inhibit the growth of fibroblasts derived from NPs. The inhibition was stronger in the case of iron-saturated LF and involved a receptor containing heparan sulfate.
The concentration of LF in saliva and nasal secretions, which is increased in pathological conditions [23, 43], might indicate a requirement for this protein to counteract inflammatory reactions. It is conceivable that different
Acknowledgments
The authors thank Ms. Zofia Sonnenberg for her excellent technical assistance. The study was supported by a statutory grant from the Polish Ministry of Education, No. 4/2008, for the Institute of Immunology, Polish Academy of Sciences, Wrocław, Poland and by the Ministry of Science and Higher Education (Poland), Grant No. 1043/B/P01/2007/33