Prolactin induces chitotriosidase gene expression in human monocyte-derived macrophages

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Prolactin induces Chitotriosidase gene expression in human monocytederived macrophages

L. Malaguarnera*, M. Musumeci*, F. Licata*, M. Di Rosa*, A. Messina*, S. Musumeci°

*Department of Biomedical Sciences, University of Catania, Italy ° Department of Pharmacology, Gynecology/Obstetrics, Pediatrics, University of Sassari and Institute of Genetic Population CNR, Alghero (SS), Italy

Corresponding author: Lucia Malaguarnera, Via Firenze, 42 95021 Acicastello – Catania, Italy. Telefax: ++39/ 95/7125739 e-mail: [email protected]

Abstract Human Chitotriosidase (Chit), a chitinolytic enzyme, is a member of the chitinase family. In human’s plasma Chit activity have been proposed as a biochemical marker of macrophage activation in several lysosomal diseases. Recently we found that Chit activity is higher in patients affected by Plasmodium falciparum malaria infection suggesting that chitotriosidase may induce an immunological response. The pituitary hormone prolactin (PRL) is a multifunctional polypeptide also produced by immune cells and represents a key component of the neuroendocrine-immune loop. The presence of PRL receptors in macrophage suggests that PRL is involved in regulating functions in these cells. Our objective in this study was to investigate the effect of PRL in human monocyte-derived macrophages (HMMs) on Chit production. Administration of PRL in HMMs was found to increase both expression and activity of Chit in a time and dose dependent manner as quantified respectively by real-time PCR and Chit activity assay. PRL-treated monocyte-derived macrophages showed also an enhanced release of superoxide anion (O-2) release. Our observations confirm that PRL regulates HMMs activation and suggest, for the first time, that it influences immune function also through the induction of Chit activity

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Introduction Human Chitotriosidase (Chit) is a member of the chitinase family with the capability to hydrolyze chitin. Chitin is present as a structural component in the coating of many living species such as the cell wall of fungi (1), the sheath of parasitic nematodes (2) and in the lining of gut of many insects (3). Human Chit exhibits a remarkable sequence homology among other chitinase from plants, bacteria, fungi, nematodes and insects (4,5). The CHIT gene is localized in the chromosome 1q31-q32 (6), the gene consists of 12 exons and spans about 20 kilobases of genomic DNA (4). Chit is mainly secreted as a 50kDa active enzyme containing a C-terminal chitin binding domain (7). In human monocyte-derived macrophages (HMMS) this protein is proteolytically processed to a C-terminally truncated 39 kDa isoform characterized by hydrolase activity and accumulated in the lysosomes (7). In addition, the 50 kDa Chit form is synthesized by neutrophilic granulocyte progenitors and stored in their granules (4, 8). A recessive inherited deficiency in Chit activity is frequently encountered in different populations. A 24base pair duplication in exon 10 results in activation of a cryptic 3’ splice site, generating a abnormally spliced mRNA with an in-frame deletion of 87 nucleotides (9). The spliced mRNA encodes an enzymatic inactive protein that lacks an internal stretch of 29 amino acids (9). This CHIT mutant allele has been found in 33-35% of Ashkenazi Jewish and Dutch individuals respectively, whereas both populations were about 6% homozygous for this allele (9). Additional studies performed in different populations confirmed the presence of the 24-base pair duplication in individuals completely deficient in enzymatic active Chit (9). The enzyme chitotriosidase is of interest for clinical reasons, since have been proposed as a biochemical marker of macrophage activation in several lysosomal diseases, particularly is a valuable diagnostic tool to monitor the efficacy of therapy in Gaucher diseases or beta-glucocerebrosidase deficiency (10), a disorder characterized by the presence of large amounts of activated, lipid-laden monocyte-derived macrophages in spleen, liver and other tissues (10). In addition it has been found that serum chit activity is significantly increased in individuals suffering from atherosclerosis disease and is related to the severity of the atherosclerotic lesion, suggesting a possible role as atherosclerotic extent marker (11-13). More modest elevations are found in plasma of patients with sarcoidosis (14). Moreover, Chit is increased in patients affected by malaria infection and in other hematological disorders where activated macrophages are involved (14-16). Prolactin (PRL), a polypeptide hormone secreted by the acidophilic cell of anterior pituitary gland, is implicated in diverse array of physiological functions such as osmoregulation, reproduction, growth and development (17).

PRL synthesis has been demonstrated in extra-pituitary tissue,

including endothelial (18), neuronal and immune cells such as lymphocytes, mononuclear cells and 3

thymocytes (17). The emerging role of PRL in immunoregulation has led to the concept of a dual function for PRL as both a circulating hormone and cytokine (18-19). Moreover, perturbation of PRL physiology has significant immunological effects in humans. Hyperprolactinemia is associated with malignant diseases and also with autoimmune diseases such as multiple sclerosis, lupus erythematosus and rheumatoid arthritis (20). PRL it is structurally related to several cytokines and its receptor (PRL-R) belongs to the superfamily of hematopoietic cytokine receptors (20), which includes receptors for IL-2 ( β and γ chain), IL-3, -4, -6, -7, -8, -9, -12, -15, GM-CSF, G-CSF, EPO, ghp 130, LIF, OSM, CNTF, MPL, GH, IFNα / β and IFNγ (21, 22). Recent evidence indicates that PRL is involved in regulating monocyte/macrophage functions, suggested also by the presence of PRL-R in these cells (23). Repeated treatment with PRL protected mice from Salmonella typhimurium infection (24). Increased phagocytosis, intracellular killing and NO release by the macrophages was suggested to be underlying mechanism of protection against microbial infections (25). O-2, NO-2 and TNF-α constitute the major cytotoxic effector molecules in macrophage-mediated killing of microbes and tumor cells (26). Our previous studies provided direct evidence that IFN-γ, TNF-α and LPS upregulate CHIT gene expression (27). Since it is known that macrophages treated with IFN-γ and TNF-α develop an increased cytocidal activity against intracellular micro-organism and tumor cells, we argued that Chit may be involved in cellular response elicited by regulatory cytokine. On the basis of these findings it can be hypothesized that Chit activity increases in response to various immunological stimuli. Most importantly PRL modulates the expression of genes crucial for leukocytes function. The aim of our study was to demonstrate a specific link between PRL and CHIT expression, analyzing both Chit activity and CHIT mRNA levels in HMMs following treatment with PRL.

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RESULTS Superoxide anion (O-2) production. To investigate the involvement of PRL in regulating HMMs activity the O-2 release was carried out by the spectrophotometric measurement of ferricytochrome c reduction. HMMs of peripheral blood were cultured for 4 days, thereafter were cultured at different times (2, 4, 8, 16 and 24 hours) and in absence or presence of different concentration (5, 10, 25, 50 and 100 ng/ml) of PRL. As shown in fig. 1 PRL induced O-2 production in a dose, and time-dependent manner. Within 4h of PRL (25 ng/ml) the production of O-2 was increased 8.68-fold over that of the control (P
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