|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Immunohistochemical evidence of lactoferrin in human embryo–fetal bone and cartilage tissues
Ieni Antonio2, Barresi Valeria, Grosso Maddalena and Tuccari Giovanni1
Department of Human Pathology, University of Messina, Messina, Italy
Lf (lactoferrin) is an 80-kDa iron-binding protein, which has been suggested to promote bone growth in murine models. In view of this, we aimed to analyse the immunohistochemical distribution of Lf in human embryonal and fetal bone and cartilaginous tissues at different gestational weeks in order to evaluate whether a role for this protein might be proposed also in human osteogenesis. Bone and cartilaginous specimens were taken at autopsy from 25 fetuses (8–34 weeks of gestation). Ten samples of human adult bone and cartilage were also submitted to the immunohistochemical procedures. Sections, 4-μm thick, were cut from formalin-fixed paraffin-embedded tissue blocks and stained with a monoclonal antibody against human Lf, following antigen retrieval procedures. Lf immunoreactivity was maily localized in the mesenchymal cells forming the periosteum as well as in chondroblasts at the eighth gestational week; a strong Lf immunoexpression in immature osteocytes and osteoblasts was noted up to the 18th gestation week, with a considerable decrease by the 24th week. No Lf expression was found in any bone area after the 30th and up to the 34th week of gestation. Our data seem to suggest an important role for Lf as a bone growth regulator in the early phases of the human endochondral ossification, with an anabolic action similar to that previously reported in cell culture lines and in animal models.
Key words: chondroblast, immature osteocyte, immunohistochemistry, lactoferrin, mesenchymal cell
Abbreviations: Lf, lactoferrin, MSCs, mesenchymal stem cells
1Dedicated to my father in the occasion of his 90th birthday.
2To whom correspondence should be addressed (email email@example.com).
In the human lifetime, bones experience slow prepubertal growth, rapid pubertal growth, balanced remodelling and, finally, bone loss (Riggs and Melton, 2002). All these steps are controlled by changes in hormones, growth factors, mechanical loading, nutrition and other unidentified factors. There are two major modes of osteogenesis. The first, called intramembranous ossification, occurs in the bones of the skull; in the second, named endochondral ossification, mesenchymal cells differentiate into cartilage, which is later replaced by bone. The regulation of cartilage calcification is essential to bone growth and modelling, although the mechanisms of calcification as well as the occurring factors are still not fully understood and identified. Recently, it has been suggested that Lf (lactoferrin) promotes bone growth in a murine model, with an anabolic action due to the stimulation of the proliferation of osteoblasts and cartilage cells in organ culture (Cornish, 2004; Naot et al., 2005; Cornish et al., 2006; Cornish and Naot, 2010).
Lf is an 80-kDa iron-binding glycoprotein, produced by many exocrine glands and largely distributed in body fluids such as tears, saliva, bile, pancreatic fluid, vaginal secretions, semen, colostrum, milk and amniotic fluid (Levay and Viljoen, 1995; Lönnerdal and Iyver, 1995). Lf acts as an iron chelator, playing a role in the host defence mechanism with bacteriostatic and bactericidal actions (Levay and Viljoen, 1995; Weinberg, 2001). Moreover, it has effects on cytokine and chemokine production (Elass et al., 2002; Kimber et al., 2002), modulation of the inflammatory response (Baveye et al., 1999) and murine embryonic development (Ward et al., 1999).
The distribution of Lf has been investigated by immunohistochemistry in a number of normal human adult tissues such as stomach, kidney, lung, pancreas, liver and bone marrow (Mason and Taylor, 1978). In addition, Lf immunoexpression has been studied in seven human fetuses from 11 to 21 weeks of gestational age by (Reitamo et al., 1981) using a rabbit polyclonal antibody, and it has been found in mononuclear cells of various organs since the 13th week of gestation onwards (Reitamo et al., 1981). As no data about Lf distribution in the embryonal and fetal bone and cartilaginous tissues are available to date, we thought it of interest to analyse the immunohistochemical pattern of Lf in these tissues during fetal development in order to verify its possible role in the growth and differentiation of the human skeleton.
2. Materials and methods
The study was approved by the Local Ethics Committee. Samples of unaffected human cartilage as well as long and flat bone tissues were taken at autopsy from 25 fetuses (ranging between 8 and 34 weeks of gestation). Specifically, nine samples from fetuses below the 13th week of gestation were retrieved following legal voluntary termination of pregnancy. In the other cases, abortion was due to abruption placentae. Normal bone and cartilaginous specimens taken at autopsy from ten adults (age: 47–82 years; mean age: 68 years), after death from vascular accidents, were also submitted to the immunohistochemical procedures. Renal tubular structures within samples of normal kidney as well as portions of parotid gland were utilized as positive controls (Giuffrè et al., 2007). Informed consent was received from the next-of-kin for adult samples and from the mothers of aborted fetuses.
All samples were routinely fixed, embedded, cut and stained with haematoxylin/eosin, Perls' Prussian Blue Ferrocyanide and von Kossa methods. The immunohistochemical procedures have been extensively reported elsewhere (Ieni et al., 2009a, 2009b). Finally, immunostained sections were examined by light microscopy by using a ×20 and ×40 objective lens and ×10 eyepiece. Two pathologists, using a double-headed microscope, performed the assessment of Lf immunostained sections on a consensus basis. The IS (intensity of staining) was graded into weak (+), moderate (++) and strong (+++).
Analytical data concerning the embryos and fetuses, in relation to Lf immunoexpression, are reported in Table 1.
Table 1 Analytical Lf immunostaining in bones of 25 fetuses
pMSCs, periosteum mesenchymal stem cells; CHBL, chondroblasts; IOST, immature osteocytes; OBL, osteoblasts; M, male; F, female.
Routinely stained haematoxylin/eosin sections exhibited a good morphology in each of the examined samples, independently from the gestational age. At the 8th week of gestation, areas of calcified cartilage in endochondral ossification, representing the newly formed bone, were identified through the Von Kossa method (Figures 1b, inset). Staining greatly increased during subsequent gestational weeks. No granular blue Perls' staining was found in any cartilage and bone districts within all analysed samples.
Lf immunolabelling was mainly localized at the cytoplasm and sometimes at the nucleus of stained elements. At the 8th week of gestation Lf immunoreactivity was found in the MSCs (mesenchymal stem cells) forming the periosteum (Figure 1a), as well as in chondroblasts within all the calcified cartilage samples (Figure 1b). These latter elements exhibited a weak to moderate Lf staining (Figure 1b), while MSCs showed a strong immunoexpression (Figure 1a). At the 12th week, a strong Lf immunoexpression was found in immature osteocytes and osteoblasts enclosed or lining the spicule as well as in chondroblasts of peripheral calcified cartilage (Figures 2a, 2b). At the 18th week, a constant increase in the number of Lf-positive osteoblasts inside the bony matrix was noted (Figure 3), but a considerable decrease of staining was encountered in these cells by the 24th and up to the 29th week of gestation. No immunostaining was seen in the mature chondrocytes between 18 and 29 gestational weeks (Figure 4). In addition, no Lf expression was found in the osteocartilagineous fetal after the 30th week and up to the 34th week, similar to the adult bone tissue samples. The osteoclast lineage was always unstained by Lf antibody.
Lf immunolocalization was evident in all ductular/acinar structures of the parotid as well as in kidney tubular structures utilized as positive controls for the immunoreactions.
In the present study, we investigated Lf immunoexpression in the bone and cartilaginous tissues from human embryos and fetuses at different gestational ages. In particular, we analysed the interval between 8 and 34 weeks of gestation, which corresponds to the increase in the differentiation of MSC progenitor cells towards chondroblast and osteoblast lineages.
In stained elements, the Lf immunolabelling was mainly found in the cytoplasm and only occasionally in the nucleus. The site of Lf immunoreactivity in both the nucleus and in the cytoplasm is not surprising, since this secretory protein has been immunohistochemically reported in the nucleus, mainly in nucleoli, and it has been considered as involved in ribosomal biogenesis (Penco et al., 2001; Tuccari et al., 2005). In relation to the spatiotemporal expression of Lf during the embryo–fetal development, we reported Lf immunoreactivity in MSCs forming the periosteum as well as in chondroblasts of all calcified cartilage samples by the 8th week of gestation. Consistently, the process of calcification of cartilage matrix starts in the diaphysis by this age of gestation. Successively, at the 12th week, Lf immunoexpression was largely present in the immature osteocytes, osteoblasts as well as in the chondroblasts of long bones. Moreover, a constant increase in the number of Lf-positive osteoblasts within the bone matrix was noted at 18 weeks, together with the evidence of calcified cartilage and osteoid. By contrast, an evident decrease in Lf immunostaining was found in the same elements after the 24th week up to the 29th of gestation. No evidence of Lf immunoreactivity was seen in the mature chondrocytes between 18 and 29 weeks. Finally, no immunoexpression of Lf was found in bone and cartilage fetal samples by the 30th and up to the 34th week, similarly to the adult bone tissue samples. The existence of a different immumohistochemical localization of Lf in relation to different stages of development has been already reported during murine embryogenesis with three distinct spatiotemporal patterns (Ward et al., 1999); in particular, it has been shown that the immunopattern of Lf is tightly regulated in preimplantation embryo, postimplantation neutrophils and epithelial cells of developing digestive and respiratory tract (Ward et al., 1999).
Although many biological functions of Lf have been addressed (Brock, 1995; Lönnerdal et al., 1995; Levay and Viljoen, 1995), the biological importance of Lf in human embryo–fetal bone and cartilage samples still remains unexplained. In experimental studies, it has been suggested that Lf acts to expand the pool of preosteoblastic cells by exerting mitogenic and anti-apoptotic effects as well as by driving differentiation of precursors to produce a more mature osteoblastic phenotype capable of promoting bone matrix deposition and mineralization (Cornish et al.,2004; Naot et al., 2005, Cornish et al., 2006, Cornish and Naot, 2010). Therefore, the appearance of Lf in human bone and cartilage of the fetal skeleton, which we observed between the 8th and the 29th week of gestation, might suggest an important role of Lf as a bone growth regulator in the early phases of endochondral ossification, thus confirming our preliminary observations in a small cohort of three cases (Ieni et al., 2009a, 2009b). The documented presence of Lf, first in MSCs and later in chondroblasts and immature osteoblasts, could be related to Lf production by these elements themselves, although this interpretation should be verified by methods other than morphological analysis. Alternatively, the immunolocalization of Lf in the abovementioned elements may not reflect an intracellular synthesis, but rather be the consequence of defective or functionally impaired Lf receptors, as previously suggested (Roiron et al., 1989; Giuffrè et al., 2007).
In the present analysis, another intriguing finding was represented by the immunohistochemical disappearance of Lf from the bone and cartilagineous embryo–fetal tissues after the 29th gestational week, similarly to the osteocartilagineous samples from adults, in which Lf is always absent. Taking into consideration the high affinity of Lf for iron, it may be suggested that rapidly dividing cells, such as fetal osteocartilagineous elements in the early period of gestation, may produce Lf in order to have a greater avalibility of iron, which takes parts in metabolic cellular processes, such as oxidative phosphorylation as well as RNA and DNA synthesis. On the other hand, the absence of Lf expression in the corresponding adult cells does not seem surprising; indeed, by the age of the 30 years, metabolic activity of bone and cartilage begins to decrease, causing a bone mass reduction. Although it is well-known that osteoblasts and chondrocytes share the same progenitor (Karsenty, 2008), the process of differentiation into different lineages is controlled by many growth factors/cytochines and not exclusively by Lf. In fact, a recent study suggested that the signalling pathway activated by Lf was not sufficient to induce osteogenic differentiation of the MG63 cells (Takayama and Mizumaki, 2009). Therefore, it could be argued that bone growth and formation may be also regulated by others effectors or signalling pathways, activated by IGF-I, BMPs, Wnt, Notch, FGFs as well as nutrition, aging and mechanical loading (Bonewald and Johnson, 2008). Finally, since it has been reported that the osteogenic activity of Lf is maintained also in Lf deglycosylated, holo and apo forms (Cornish et al., 2006), we cannot exclude the presence of these different forms of Lf in osteocartilagineous cells during the later period of pregnancy, which could not be revealed by the monoclonal antibody utilized by us.
Since Lf immunoexpression has been reported in several human tumours (Barresi and Tuccari, 1984; Tuccari and Barresi, 1985; Tuccari et al., 1989; Tuccari et al., 1992; Tuccari et al., 1997; Tuccari et al., 2005; Giuffrè et al., 2006, 2007) including osteocartilagineous neoplasms (Ieni et al., 2009a, 2009b), the reported role for Lf as a tumour suppressor by inhibiting neoplastic proliferation (Zhou et al., 2008) should be considered with caution. Present data concerning the Lf immunostaining in early phases of skeleton development may suggest that Lf behaves as an oncofetal antigen in neoplastic human pathology, although this requires further investigations.
The immunohistochemical presence of Lf was demonstrated in mesenchymal cells, chondroblasts and immature osteocytes starting from the 8th until the 29th gestation weeks of human fetuses. No immunoexpression of Lf was encountered in bone and cartilage fetal samples from the 30th to the 34th gestation weeks, similarly to that shown in adults. Our data should suggest an important role for Lf as a bone growth regulator in early phases of endochondral ossification, although other effectors or signalling pathways may be activated during the later period of the pregnancy.
Antonio Ieni and Giovanni Tuccari were responsible of the conception and design. Maddalena Grosso was in charge of the provision of study materials or patients. Antonio Ieni, Maddalena Grosso and Giovanni Tuccari were involved in the collection and assembly of data. Antonio Ieni, Valeria Barresi and Giovanni Tuccari were responsible for the data analysis and interpretation. Antonio Ieni and Giovanni Tuccari wrote the manuscript. Antonio Ieni, Valeria Barresi, Maddalena Grosso and Giovanni Tuccari were in charge of the final approval of the manuscript.
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Baveye, S, Elass, E, Mazurier, J, Spik, G and Legrand, D (1999) Lactoferrin: a multifunctional glycoprotein involved in the modulation of the inflammatory process. Clin Chem Lab Med 37, 281-6
Cornish, J, Callon, KE, Naot, D, Palmano, KP, Banovic, T and Bava, U (2004) Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo. Endocrinology 145, 366-74
Cornish, J, Palmano, K, Callon, KE, Watson, M, Lin, JM and Valenti, P (2006) Lactoferrin and bone; structure–activity relationships. Biochem Cell Biol 84, 297-302
Elass, E, Masson, M, Mazurier, J and Legrand, D (2002) Lactoferrin inhibits the lipopolysaccharide-induced expression and proteoglycans-binding ability of interleukin-8 in human endothelial cells. Infect Immun 70, 1860-6
Giuffrè, G, Arena, F, Scarfì, R, Simone, A, Todaro, P and Tuccari, G (2006) Lactoferrin immunoexpression in endometrial carcinomas: relationships with sex steroid hormone receptors (ER and PR), proliferation indices (Ki-67 and AgNOR) and survival. Oncol Rep 16, (2), 257-63
Ieni, A, Barresi, V, Grosso, M, Rosa, MA and Tuccari, G (2009a) Lactoferrin immuno-expression in human normal and neoplastic bone tissue. J Bone Miner Metab 27, 364-71
Kimber, I, Cumberbatch, M, Dearman, RJ, Headon, DR, Bhushan, M and Griffiths, CE (2002) Lactoferrin: influences on Langerhans cells, epidermal cytokines, and cutaneous inflammation. Biochem Cell Biol 80, 103-7
Penco, S, Scarfi, S, Giovine, M, Damonte, G, Millo, E and Villaggio, B (2001) Identification of an import signal for, and the nuclear localization of, human lactoferrin. Biotechnol Appl Biochem 34, 151-9
Riggs, BL and Melton, LJ (2002) Bone turnover matters: the raloxifene treatment paradox of dramatic decreases in vertebral fractures without commensurate increases in bone density. J Bone Miner Res 17, 11-4
Roiron, D, Amouric, M, Marvaldi, J and Figarella, C (1989) Lactoferrin-binding sites at the surface of HT29-D4 cells. Comparison with transferrin. Eur J Biochem 186, 367-73
Takayama, Y and Mizumachi, K (2009) Effect of lactoferrin-embedded collagen membrane on osteogenic differentiation of human osteoblast-like cells. J Biosci Bioeng 107, 191-5
Tuccari, G and Barresi, G (1985) Immunohistochemical demonstration of lactoferrin in follicular adenomas and thyroid carcinomas. Virchows Arch A Pathol Anat Histopathol 406, 67-74
Tuccari, G, Rizzo, A, Crisafulli, C and Barresi, G (1992) Iron-binding proteins in human colorectal adenomas and carcinomas: an immunocytochemical investigation. Histol Histopathol 7, 543-7
Tuccari, G, Rossiello, R and Barresi, G (1997) Iron binding proteins in gallbladder carcinomas. An immunocytochemical investigation. Histol Histopathol 12, 671-6
Tuccari, G, Giuffrè, G, Scarfì, R, Simone, A, Todaro, P and Barresi, G (2005) Immunolocalization of lactoferrin in surgically resected pigmented skin lesions. Eur J Histochem 49, 33-8
Ward, PP, Mendoza-Meneses, M, Mulac-Jericevic, B, Cunningham, GA, Saucedo-Cardenas, O and Teng, CT (1999) Restricted spatiotemporal expression of lactoferrin during murine embryonic development. Endocrinology 140, 1852-60
Zhou, Y, Zeng, Z, Zhang, W, Xiong, W, Wu, M and Tan, Y (2008) Lactotransferrin: a candidate tumor suppressor-deficient expression in human nasopharyngeal carcinoma and inhibition of NPC cell proliferation by modulating the mitogen-activated protein kinase pathway. Int J Cancer 123, 2065-72
Received 23 October 2009/21 April 2009; accepted 5 May 2010
Published as Cell Biology International Immediate Publication 5 May 2010, doi:10.1042/CBI20090358
© The Author(s) Journal compilation © 2010 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)