Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response

Macrophage-derived secretory leukocyte protease inhibitor (SLPI) can be induced locally as well as systemically in response to microbial products such as LPS and lipotechoic acid. It is not known whether phagocytosis of apoptotic cells, an essential function of macrophages, can regulate expression and secretion of SLPI. In this study, we report that exposure of peritoneal macrophages of BALB/c mice or murine macrophage cell lines RAW264.7 and J774.1 to apoptotic target cells induced an elevation in SLPI secretion. Secreted SLPI retained its antichymotrypsin activity. SLPI expression in thymuses from BALB/c mice that had been injected with anti-CD3 Ab to induce apoptosis of thymocytes was also elevated both at the mRNA and protein levels. Colchicine, a microtubular inhibitor, blocked the internalization of apoptotic cells by macrophages but not SLPI secretion, suggesting that surface recognition of apoptotic cells is sufficient for the induction of SLPI. Exposure of RAW264.7 cells to apoptotic CTLL-2 cells induced both SLPI and TNF-alpha, and addition of IFN-gamma inhibited SLPI but augmented TNF-alpha production. Transfection of either the secreted or a nonsecreted form of SLPI into RAW264.7 cells led to suppression of TNF-alpha production in response to apoptotic cells. Thus, macrophages secrete an increased amount of SLPI when encountering apoptotic cells, which may help to attenuate potential inflammation during clearance of these cells.     

Regulation of SLPI and elafin release from bronchial epithelial cells by neutrophil defensins

Secretory leukocyte proteinase inhibitor (SLPI) is a serine proteinase inhibitor that is produced locally in the lung by cells of the submucosal bronchial glands and by nonciliated epithelial cells. Its main function appears to be the inhibition of neutrophil elastase (NE). Recently, NE was found to enhance SLPI mRNA levels while decreasing SLPI protein release in airway epithelial cells. Furthermore, glucocorticoids were shown to increase both constitutive and NE-induced SLPI mRNA levels. In addition to NE, stimulated neutrophils also release alpha-defensins. Defensins are small, antimicrobial polypeptides that are found in high concentrations in purulent secretions of patients with chronic airway inflammation. Like NE, defensins induce interleukin-8 production in airway epithelial cells. This induction is sensitive to inhibition by the glucocorticoid dexamethasone and is prevented in the presence of alpha(1)-proteinase inhibitor. The aim of the present study was to investigate the effect of defensins on the production of SLPI and the related NE inhibitor elafin/SKALP in primary bronchial epithelial cells (PBECs). Defensins significantly increase SLPI protein release by PBECs in a time- and dose-dependent fashion without affecting SLPI mRNA synthesis. In the presence of alpha(1)-proteinase inhibitor, the defensin-induced SLPI protein release is further enhanced, but no effect was observed on SLPI mRNA levels. Dexamethasone did not affect SLPI protein release from control or defensin-treated PBECs. In addition, we observed a constitutive release of elafin/SKALP by PBECs, but this was not affected by defensins. The present results suggest a role for defensins in the dynamic regulation of the antiproteinase screen in the lung at sites of inflammation.     

Anti‐tumor effect of SLPI on mammary but not colon tumor growth

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that was related to cancer development and metastasis dissemination on several types of tumors. However, it is not known the effect of SLPI on mammary and colon tumors. The aim of this study was to examine the effect of SLPI on mammary and colon tumor growth. The effect of SLPI was tested on in vitro cell apoptosis and in vivo tumor growth experiments. SLPI over‐expressing human and murine mammary and colon tumor cells were generated by gene transfection. The administration of murine mammary tumor cells over‐expressing high levels of SLPI did not develop tumors in mice. On the contrary, the administration of murine colon tumor cells over‐expressing SLPI, developed faster tumors than control cells. Intratumoral, but not intraperitoneal administration of SLPI, delayed the growth of tumors and increased the survival of mammary but not colon tumor bearing mice. In vitro culture of mammary tumor cell lines treated with SLPI, and SLPI producer clones were more prone to apoptosis than control cells, mainly under serum deprivation culture conditions. Herein we demonstrated that SLPI induces the apoptosis of mammary tumor cells in vitro and decreases the mammary but not colon tumor growth in vivo. Therefore, SLPI may be a new potential therapeutic tool for certain tumors, such as mammary tumors. J. Cell. Physiol. 228: 469–475, 2013. © 2012 Wiley Periodicals, Inc.     

Suppression of macrophage responses to bacterial lipopolysaccharide (LPS) by secretory leukocyte protease inhibitor (SLPI) is independent of its anti-protease function

Secretory leukocyte protease inhibitor (SLPI), a potent serine protease inhibitor, has been shown to suppress macrophage responses to bacterial lipopolysaccharide (LPS). SLPI contains two topologically superimposable domains. Its C-terminal domain binds and inhibits target proteases. It is not clear whether SLPI's anti-protease function plays a role in the LPS-inhibitory action of SLPI. Four single amino acid substitution mutants of SLPI, M73G, M73F, M73E and M73K, were generated. Wild type SLPI is a potent inhibitor of chymotrypsin and elastase. Mutants M73G and M73F selectively lost inhibitory function towards chymotrypsin and elastase, respectively, whereas mutants M73K and M73E inhibited neither elastase nor chymotrypsin. Macrophage cell lines were established from RAW264.7 cells to stably express each SLPI mutant. Expression of the SLPI protease inhibition mutants suppressed NO and TNF production in response to LPS in a similar fashion as wild type SLPI. Expression of truncated forms of SLPI, containing only its N-terminus or its C-terminus, was similarly sufficient to confer inhibition of LPS responses. Thus, the LPS-inhibitory action of SLPI is independent of its anti-protease function.     

Secretory leucoprotease inhibitor prevents lipopolysaccharide-induced IkappaBalpha degradation without affecting phosphorylation or ubiquitination

Secretory leucoprotease inhibitor (SLPI) is a non-glycosylated protein produced by epithelial cells, macrophages, and neutrophils and was initially identified as a serine protease inhibitor of the neutrophil proteases elastase and cathepsin G. In addition to its antiprotease activity, SLPI has been shown to exhibit anti-inflammatory properties including down-regulation of tumor necrosis factor-alpha expression by lipopolysaccharide (LPS) in monocytes, inhibition of NF-kappaB activation by IgG immune complexes in a rat model of acute lung injury, and prevention of human immunodeficiency virus infectivity in monocytic cells via as yet unidentified mechanisms. In this report we have shown that SLPI prevents LPS-induced NF-kappaB activation by inhibiting degradation of IkappaBalpha without affecting the LPS-induced phosphorylation and ubiquitination of IkappaBalpha. We have also demonstrated that SLPI prevents LPS-induced interleukin-1 receptor-associated kinase and IkappaBbeta degradation. In addition, we have demonstrated that oxidized SLPI, a variant of SLPI that has diminished antiprotease activity, cannot prevent LPS-induced NF-kappaB activation or Inhibitor kappaB alpha/beta degradation indicating that the anti-inflammatory effect of SLPI on the LPS-signaling pathway is dependent on its antiprotease activity. These results suggest that SLPI may be inhibiting proteasomal degradation of NF-kappaB regulatory proteins, an effect that is dependent on the antiprotease activity of SLPI.     

Secretory leukocyte protease inhibitor mediates proliferation of human endometrial epithelial cells by positive and negative regulation of growth-associated genes

Secretory leukocyte protease inhibitor (SLPI) inhibits chymotrypsin, trypsin, elastase, and cathepsin G. This protein also exhibits proliferative effects, although little is known about the molecular mechanisms underlying this activity. We have generated SLPI-ablated epithelial sublines by stably transfecting the Ishikawa human endometrial cell line with an antisense human SLPI RNA expression vector. We demonstrate a positive correlation between cellular SLPI production and proliferation. We further show that Ishikawa sublines expressing low to undetectable SLPI have correspondingly increased and decreased expression, respectively, of transforming growth factor-beta 1 and cyclin D1 genes, relative to parental cells. SLPI selectively increased cyclin D1 gene expression, with the effect occurring in part at the level of promoter activity. Cellular SLPI levels negatively influenced the anti-proliferative and pro-apoptotic insulin-like growth factor-binding protein-3 expression. We also identified lysyl oxidase, a phenotypic inhibitor of the ras oncogenic pathway and a tumor suppressor, as SLPI-repressed gene, whose expression is up-regulated by transforming growth factor-beta1. Our results suggest that SLPI acts at the node(s) of at least three major interacting growth inhibitory pathways. Because expression of SLPI is generally high in epithelial cells exhibiting abnormal proliferation such as in carcinomas, SLPI may define a novel pathway by which cellular growth is modulated.     

Secretory leukocyte proteinase inhibitor-competent DNA deposits are potent stimulators of plasmacytoid dendritic cells: implication for psoriasis

Secretory leukocyte proteinase inhibitor (SLPI) is a well-established inhibitor of serine proteases such as human neutrophil elastase (HNE) and a NF-κB regulatory agent in immune cells. In this paper, we report that SLPI plays a previously uncharacterized role in regulating activation of plasmacytoid dendritic cells (pDCs). As the main source of IFN type I (IFNI), pDCs are crucial contributors to inflammatory and likely wound-healing responses associated with psoriasis. The mechanisms responsible for activation of pDCs in psoriatic skin are therefore of substantial interest. We demonstrate that in lesional skin of psoriasis patients, SLPI together with its enzymatic target HNE and DNA, is a component of neutrophil extracellular traps (NETs). Whereas SLPI(+) neutrophils and NETs were found to colocalize with pDCs in psoriatic skin, a mixture of SLPI with neutrophil DNA and HNE induced a marked production of IFNI by pDCs. IFNI synthesis by stimulated pDCs was dependent on intracellular DNA receptor TLR9. Thus, SLPI may contribute to psoriasis by enabling pDCs to sense extracellular DNA and produce IFNI.     

Identification of SLPI (secretory leukocyte protease inhibitor) in human mast cells using immunohistochemistry and in situ hybridisation.

Recently interest has been focused on secretory leucocyte protease inhibitor (SLPI) and its role in immediate hypersensitive reactions, possibly by inhibiting mast cell chymase. The purpose of this investigation was to show whether or not SLPI is produced in mast cells. Double-immunolabelling revealed that SLPI coexists with mast cell tryptase (60%) and chymase (37%). On the other hand, in situ hybridisation studies demonstrated the expression of SLPI mRNA in all mast cells. The differences in results can be attributed to the fact that in situ hybridisation is a more sensitive method than immunohistochemistry. Hence, we conclude that SLPI is produced in human tonsillar mast cells.     

Increased Secretory Leukocyte Protease Inhibitor (SLPI) Production by Highly Metastatic Mouse Breast Cancer Cells

The precise molecular mechanisms enabling cancer cells to metastasize from the primary tumor to different tissue locations are still largely unknown. Secretion of some proteins by metastatic cells could facilitate metastasis formation. The comparison of secreted proteins from cancer cells with different metastatic capabilities in vivo might provide insight into proteins involved in the metastatic process. Comparison of the secreted proteins from the mouse breast cancer cell line 4T1 and its highly metastatic 4T1.2 clone revealed a prominent differentially secreted protein which was identified as SLPI (secretory leukocyte protease inhibitor). Western blotting indicated higher levels of the protein in both conditioned media and whole cell lysates of 4T1.2 cells. Additionally higher levels of SLPI were also observed in 4T1.2 breast tumors in vivo following immunohistochemical staining. A comparison of SLPI mRNA levels by gene profiling using microarrays and RT-PCR did not detect major differences in SLPI gene expression between the 4T1 and 4T1.2 cells indicating that SLPI secretion is regulated at the protein level. Our results demonstrate that secretion of SLPI is drastically increased in highly metastatic cells, suggesting a possible role for SLPI in enhancing the metastatic behavior of breast cancer cell line 4T1.     

SLPI suppresses hepatocellular carcinoma progression via endoplasmic reticulum stress induced apoptosis

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Secretory leukocyte protease inhibitor (SLPI) has been reported to function as a regulatory factor in several cancers. However, its biological functions and underlying mechanisms in HCC remain to be uncovered. Here, we aimed to explore the effect of SLPI in HCC. In our study, we found that the mRNA and protein expression levels of SLPI were significantly down-regulated in HCC tissues and hepatoma cell lines and low level of SLPI predicted worse survival in our HCC cohorts. In term of function, silencing of SLPI markedly promoted whereas overexpression SLPI suppressed proliferation, migration and invasion capabilities of HCC cells in vitro, and ectopic expression of SLPI inhibited the tumorigenicity of HCC cells in vivo. Mechanistic studies demonstrated that SLPI played a protective role in HCC progression via activating endoplasmic reticulum stress (ER stress)-mediated apoptosis of hepatoma cells, which could be regulated by MAPK signaling pathways. In summary, our findings highlight that SLPI could serve as a potential prognostic biomarker and putative tumor suppressor by enhancing ER stress-induced apoptosis in HCC cells mediated by MAPK signaling pathways, which provides new insights into promising therapeutic targets for HCC treatment.     

Potent antimycobacterial activity of mouse secretory leukocyte protease inhibitor

Secretory leukocyte protease inhibitor (SLPI) has multiple functions, including inhibition of protease activity, microbial growth, and inflammatory responses. In this study, we demonstrate that mouse SLPI is critically involved in innate host defense against pulmonary mycobacterial infection. During the early phase of respiratory infection with Mycobacterium bovis bacillus Calmette-Guérin, SLPI was produced by bronchial and alveolar epithelial cells, as well as alveolar macrophages, and secreted into the alveolar space. Recombinant mouse SLPI effectively inhibited in vitro growth of bacillus Calmette-Guérin and Mycobacterium tuberculosis through disruption of the mycobacterial cell wall structure. Each of the two whey acidic protein domains in SLPI was sufficient for inhibiting mycobacterial growth. Cationic residues within the whey acidic protein domains of SLPI were essential for disruption of mycobacterial cell walls. Mice lacking SLPI were highly susceptible to pulmonary infection with M. tuberculosis. Thus, mouse SLPI is an essential component of innate host defense against mycobacteria at the respiratory mucosal surface.     

SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes,in which Galectin4 plays an important role

ABSTRACT

To elucidate the underlying mechanism of secretory leukocyte protease inhibitor (SLPI)-induced cell migration, we compared SLPI-deleted human gingival carcinoma Ca9-22 (ΔSLPI) cells and original (wild-type: wt) Ca9-22 cells using several microscopic imaging methods and gene expression analysis. Our results indicated reduced migration of ΔSLPI cells compared to wtCa9-22 cells. The lamellipodia/dorsal ruffles were smaller and moved slower in ΔSLPI cells compared to wtCa9-22 cells. Furthermore, well-developed intermediate filament bundles were observed at the desmosome junction of ΔSLPI cells. In addition, Galectin4 was strongly expressed in ΔSLPI cells, and its forced expression suppressed migration of wtCa9-22 cells. Taken together, SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.     

Secretory leukocyte protease inhibitor: inhibition of human immunodeficiency virus-1 infection of monocytic THP-1 cells by a newly cloned protein

The ability of the salivary protein, secretory leukocyte protease inhibitor (SLPI), to inhibit human immunodeficiency virus-1 (HIV-1) infection in vitro has been reported previously and has led to the suggestion that SLPI may be partially responsible for the low oral transmission rate of HIV-1. However, results contradictory to these findings have also been published. These discrepancies can be attributed to a number of factors ranging from the variability of macrophage susceptibility to HIV infection to the quality of commercially available preparations of SLPI. To resolve these differences and to study further the potential anti-HIV-1 activity of SLPI, the purified and re-folded protein, expressed from a synthetic gene, was examined using human monocytic THP-1 cells. This newly cloned SLPI reduced HIV-1(Ba-L) infection in differentiated THP-1 cells, in contrast to the results observed when using commercially available preparations of SLPI. Interestingly, while the two proteins displayed different anti-HIV effects they had comparable anti-protease activity. The identification of the THP-1 cell line as a system that supports HIV replication, which can be inhibited by a preparation of SLPI now available in large quantities, sets the stage for a thorough investigation of the molecular and structural basis for the anti-HIV activity of SLPI.     

IgE-mediated histamine release from nasal mucosa is inhibited by SLPI (secretory leukocyte protease inhibitor) to the level of spontaneous release.

The secretory leukocyte protease inhibitor (SLPI) is a low-molecular-weight inhibitor of proteases, such as elastase and cathepsin G which are released from leukocytes during phagocytosis. The purpose of this study was to determine whether or not SLPI is able to inhibit IgE-mediated histamine release. Nasal mucosa from 11 test subjects without atopic disposition was used for this in vitro study. We found that SLPI inhibited histamine release in a dose-dependent way but was without influence on the spontaneous release.