Production Of Secretory Leukocyte Protease Inhibitor Research Articles

Plasma concentrations of secretory leukocyte protease inhibitor (SLPI) differ depending on etiology and severity in community-onset bloodstream infection

The severity of bloodstream infections (BSI) depends on pathogen, source, and host factors. Secretory leukocyte protease inhibitor (SLPI) counteracts tissue damage, balances inflammation, and is increased in pneumonia and sepsis. We aimed to evaluate whether SLPI production differs depending on etiology, disease severity, and sex in BSI and to correlate SLPI with markers of inflammation and immunosuppression. Of the adult patients with BSI, 109 were included and sampled repeatedly, from hospital admission through day 28. Controls (blood donors) were sampled twice. SLPI in plasma was measured with enzyme-linked immunosorbent assay (ELISA) technique. Streptococcus pneumoniae and Staphylococcus aureus etiology were associated with higher SLPI than Escherichia coli on days 1–2 and 3. On day 1–2, subjects with sepsis had higher SLPI concentrations than those with non-septic BSI. Pneumonia was associated with higher SLPI than a non-pulmonary source of infection. SLPI co-varied with inflammatory markers. SLPI concentrations did not differ with regard to sex in the full cohort, but men with pneumonia had higher SLPI than women on day 1–2. S. pneumoniae and S. aureus BSI were associated with higher SLPI, when compared to E. coli. Severity and pneumonia, as well as male sex in the pneumonia sub-cohort, were factors independently associated with higher SLPI.

Regulation of neutrophil elastase-induced MUC5AC expression by nuclear factor erythroid-2 related factor 2 in human airway epithelial cells.

Mucin 5AC (MUC5AC) is one of the main airway mucins implicated in pulmonary diseases with mucous hypersecretion. Neutrophil elastase (NE), a serine protease released by neutrophils, is known to induce MUC5AC synthesis by increasing reactive oxygen species (ROS) generation. Nuclear factor erythroid-2 related factor 2 (Nrf2), a basic-region leucine-zipper transcription factor, is believed to protect against ROS damage by activating a series of antioxidant enzymes; Nrf2 is also reported to reduce NE activity. The aim of our study was to examine the relationship between Nrf2 expression and NE-induced MUC5AC production. We used a small interfering RNA to inhibit Nrf2 expression. The Nrf2 gene and protein expressions were assessed by quantitative real-time polymerase chain reaction, and Western blotting, respectively. The ROS generation was examined using a kit. The expression of MUC5AC was assessed using enzyme-linked immunosorbent assay. The results showed that NCI-H292 epithelial cells, in which the Nrf2 gene expression repressed, were highly predisposed to NE stimulation, with marked exacerbation of ROS generation and reduced secretory leukocyte protease inhibitor production, resulting in high MUC5AC expression. Pretreatment with the potent Nrf2 activator sulforaphane had the reverse effect. These results demonstrate that Nrf2 is a novel nuclear factor involved in down-regulating MUC5AC synthesis by inhibiting ROS generation and augmenting proteinase inhibitor production.

Hypoxia down-regulates expression of secretory leukocyte protease inhibitor in bronchial epithelial cells via TGF-β1.

BackgroundSecretory leukocyte protease inhibitor (SLPI) is a protein with anti-protease and antimicrobial properties that is constitutively secreted from the airway epithelium. The importance of maintaining a balance between proteases and anti-proteases, and robust innate defence mechanisms in the airways, is exemplified by inflammatory lung conditions such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Both conditions present with a high protease burden in the airways which leads to tissue destruction. These patients also have an impaired innate immune system in the lungs with bacterial colonization and frequent airway infections. Moreover, both diseases are associated with airway hypoxia due to inflammation and mucus plugs. The aim of the present study was to investigate the role of hypoxia on SLPI production from the airway epithelium.MethodsPrimary human bronchial epithelial cells were grown in sub-immersed cultures or as differentiated epithelium in air liquid interface cultures. Cells were incubated at 21% O2 (normoxia) or 1% O2 (hypoxia), and the release of SLPI was analysed with ELISA. RT-PCR was used to study the expression of SLPI and transforming growth factor β1 (TGF-β1).ResultsHypoxia decreased the constitutive production of SLPI by bronchial epithelial cells. The multifunctional cytokine TGF-β1, which is known to affect SLPI expression, showed increased expression in hypoxic bronchial epithelial cells. When bronchial epithelial cells were exposed to exogenous TGF-β1 during normoxia, the SLPI production was down-regulated. Addition of TGF-β1-neutralizing antibodies partially restored SLPI production during hypoxia, showing that TGF-β1 is an important regulator of SLPI during hypoxic conditions.ConclusionsThe mechanism described here adds to our knowledge of the pathogenesis of severe pulmonary diseases associated with hypoxia, e.g. COPD and CF. The hypoxic down-regulation of SLPI may help explain the protease/anti-protease imbalance associated with these conditions and vulnerability to airway infections. Furthermore, it provides an interesting target for the treatment and prevention of exacerbation in these patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12890-015-0016-0) contains supplementary material, which is available to authorized users.

Compartmentalized secretory leukocyte protease inhibitor expression and hormone responses along the reproductive tract of postmenopausal women

Immunity and hormonal responses in the reproductive tissues of postmenopausal women are poorly understood. Secretory leukocyte protease inhibitor (SLPI), a multifunctional antimicrobial protein expressed at mucosal surfaces, is thought to play a key role in infectious and inflammatory contexts. The aim of this study was to measure SLPI production along the female reproductive tract in postmenopausal women with and without hormonal treatment. We additionally quantified estrogen receptor alpha (ERα) and progesterone receptor A (PRA) in these tissues. Expression of SLPI was decreased in the vagina and ectocervix of women under hormonal treatment. Endocervical ERα mRNA expression was increased while this did not reach significance at the protein level. SLPI expression in the endometrium was not influenced by hormonal treatment. We observed attenuated ERα expression in the cervix and endometrium of hormonally treated women, whereas vaginal expression was increased. PRA expression was augmented in the cervix and endometrium and unchanged in the vagina. Taken together, our results indicate that hormonal responses and receptor expression are differentially regulated in vaginal tissue compared with the cervix and endometrium.

The Epithelium Takes Center Stage in Allergic Keratoconjunctivitis

To investigate the role of the epithelium in severe allergic conjunctivitis. We first investigated the expression of protease-activated receptors (PARs) in cultured human conjunctival epithelial cells and fibroblasts by reverse transcription-polymerase chain reaction. Next, we examined whether mite allergen-stimulated cells release chemokines and whether physiological protease inhibitors such as secretory leukocyte protease inhibitor (SLPI) and α1-antitrypsin can inhibit their production. We also looked at the expression of thymic stromal lymphopoietin (TSLP) in giant papillae of patients with vernal keratoconjunctivitis and examined whether the as Toll-like receptor 3 ligand polyinosinic:polycytidylic acid (poly I:C) can induce expression of TSLP in cultured human conjunctival epithelial cells. PAR 1, PAR2, and PAR3 were expressed in cultured human conjunctival epithelial cells and fibroblasts at mRNA level. These epithelial cells released interleukin (IL) 6 and IL-8, with an upregulation in their gene expression, in response to the serine protease activity of mite allergens. This response was inhibited by SLPI and α1-antitrypsin. Transforming growth factor β1 decreased the production of SLPI in corneal and conjunctival epithelial cells. TSLP was expressed in giant papillae epithelium in patients with vernal keratoconjunctivitis at mRNA and protein levels. Poly I:C induced expression of TSLP in cultured conjunctival epithelial cells at mRNA level. Costimulation with TSLP and IL-33 had a synergistic effect for IL-13 mRNA expression in cultured human mast cells. Imbalance between protease of mite allergens and innate protease inhibitors of the epithelium may induce inflammation and disrupt barrier function. Viral infection may induce expression of TSLP via Toll-like receptors and release IL-33 by necrosis. These phenomena promote excessive allergic reactions; hence, the epithelium takes "center stage" in allergic conjunctivitis.

Regulation of Neutrophil Elastase-Induced MUC5AC Expression by Nuclear Factor Erythroid-2 Related Factor 2 in Human Airway Epithelial Cells

Mucin 5AC (MUC5AC) is one of the main airway mucins implicated in pulmonary diseases with mucous hypersecretion. Neutrophil elastase (NE), a serine protease released by neutrophils, is known to induce...

The mouth: a gateway or a trap for HIV?

Mucosal sites represent the primary routes of HIV transmission, yet the oral mucosa appears uniquely resistant to HIV-1 infection. Since the oral cavity is not conducive to either infection or transmission of HIV, characterizing the responsible resistance factors, particularly components of innate immunity, and their mechanisms of action may identify new opportunities for interference with viral acquisition at other mucosal sites. Although many of the innate immune factors described to date exhibit both antibacterial and antiviral activities, molecules that inhibit HIV-1 are largely HIV-1 specific. Nonetheless, if these oral innate factors can be identified, characterized, and harnessed, they may be useful in protection and/or intervention in acute HIV-1 infection. Endogenous molecules acting independently or synergistically, given their lack of substantive side effects, may represent an alternative novel approach for reducing HIV risk and suppressing infection. While HIV infection via breast milk does involve oral trafficking and is an important area of study, it will only be briefly discussed here. The major emphasis of this article is on the oral cavity and salivary substances that can influence HIV infection Epidemiology of oral transmission of HIV More than twenty-five years into the global HIV/AIDS epidemic, it is well established that in addition to blood, the reproductive and/or rectal mucosae represent the primary portals of HIV entry in adults. There is less definitive evidence regarding whether other mucosal sites serve as viral conduits and in particular, uncertainty remains as to the possibility of transmission of HIV-1 via the oral entrance to the gastrointestinal (GI) tract. This is not a trivial concern, since despite the successes of combination therapy for HIV/AIDS, infection rates remain high, compounded by persistence of latent viral reservoirs, antiretroviral drug resistance, and the lack of an effective vaccine or potential cure. Although it is often accepted that oral transmission is of little or no consequence, we have failed to capitalize on that information to identify the mechanism(s) of protection at the oral mucosal surface. These mechanisms could provide clues for novel strategies of prevention/intervention at other more vulnerable viral entry sites. Early in the epidemic, evidence of the presence of HIV-1 in the oral cavity suggested the possibility of transmissibility via this route [1, 2]. Whereas in some early studies [3–7], oral transmission could not be established, a few studies implicated this route as a potential, even likely mode of transmission (e.g., [8]). In larger cohorts of individuals and in well-designed studies to monitor oral transmission, the rarity of HIV-1 infection by this route was substantiated [9]. Nonetheless, potential shortcomings in these studies, including accurately documenting the definitive route of infection, underscore our lack of certainty regarding the relative risk of oral transmission. (Reviews: [10, 11]). The low frequency of documented oral transmission of HIV-1 could reflect low titers of infectious virus in saliva. While high levels of HIV-1 RNA and antigens can routinely be measured in oral fluids, infectious virus was not detected in early studies attempting to isolate infectious virus from saliva [12, 13] or more recently in large cohort studies, despite plasma viremia [14, 15].

Human Immunodeficiency Virus Type 1 Stimulates the Expression and Production of Secretory Leukocyte Protease Inhibitor (SLPI) in Oral Epithelial Cells: a Role for SLPI in Innate Mucosal Immunity

The innate immune response is a key barrier against pathogenic microorganisms such as human immunodeficiency virus type 1 (HIV-1). Because HIV-1 is rarely transmitted orally, we hypothesized that oral epithelial cells participate in the innate immune defense against this virus. We further hypothesized that secretory leukocyte protease inhibitor (SLPI), a 12-kDa mucosal antiviral protein, is a component of the host immune response to this virus. Here we demonstrated constitutive expression and production of SLPI in immortalized human oral keratinocytes. Brief exposure of cells to HIV-1 BaL and HXB2 significantly increased SLPI mRNA and protein production compared to that in mock-exposed cells (P < 0.01), as evaluated by real-time quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay. HIV-1-mediated stimulation of SLPI occurred at the transcriptional level, was dose and time dependent, was elicited by heat-inactivated and infectious viruses, and did not depend on cellular infection. Experiments with purified retroviral proteins showed that the stimulatory effect was induced specifically by external envelope glycoproteins from HIV-1 and simian immunodeficiency virus. SLPI responsiveness to HIV-1 was also observed in an unrelated oral epithelial cell line and in normal (nonimmortalized) human oral epithelial cells isolated from healthy uninfected gingival tissues. In this first report of SLPI regulation by HIV-1, we show that the expression and production of the antimicrobial and anti-inflammatory protein can be stimulated in oral epithelial cells by the virus through interactions with gp120 in the absence of direct infection. These findings indicate that SLPI is a component of the oral mucosal response to HIV-1.

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.

Effect of menstrual status on antibacterial activity and secretory leukocyte protease inhibitor production by human uterine epithelial cells in culture.

The objective of this study was to examine the production of antibacterial factor(s) by uterine epithelial cells from pre- and postmenopausal women. Apical rinses from polarized epithelial cells recovered from women at the proliferative and secretory stages of the menstrual cycle were equally effective in killing Staphylococcus aureus and Escherichia coli, but those from postmenopausal women were not. Secretory leukocyte protease inhibitor (SLPI) concentrations of apical washes from premenopausal women were significantly higher than those obtained from postmenopausal women. SLPI production correlated with bactericidal activity with respect to menstrual status and time in culture. Anti-SLPI antibody significantly decreased bactericidal activity of premenopausal epithelial cell rinses. The endometrial epithelial cell line HEC-1A did not have a bactericidal effect, nor did it produce SLPI. In contrast, HEC-1B cells produced SLPI and a factor that inhibited bacterial growth. These results indicate that menstrual status (pre- vs. postmenopausal) influences the production of SLPI and bactericidal activity by uterine epithelial cells.

Salivary Concentration of Secretory Leukocyte Protease Inhibitor, an Antimicrobial Protein, Is Decreased with Advanced Age

Background: Secretory leukocyte protease inhibitor (SLPI) exhibits antimicrobial activities that, in addition to other well-characterized proteins such as lysozyme and lactoferrin, is thought to play a critical role in mucosal defenses. Although elderly individuals are particularly susceptible to mucosal infections, salivary production of SLPI has not been assessed in an aged cohort. Objectives: Hypothesizing that oral SLPI concentrations are reduced with advanced age, this cross-sectional study compared SLPI concentrations to concentrations of lysozyme, lactoferrin and total protein in unstimulated salivary secretions of healthy, community-dwelling 79+-year-old and younger adults. Methods: Study participants were 45 non-hospitalized dentate adults aged 79–89 (23 elderly) or 21–51 years (22 non-elderly). Home-based interviews and clinical examinations determined dentate status and confirmed the absence of dentures, oral mucosal disease, anti-infective medication use, irradiation therapy for head and neck cancer and self-perceived xerostomia. Whole unstimulated saliva was collected from all subjects and analyzed for antimicrobial protein concentration by enzyme-linked immunosorbent assay and for total protein content by the bicinchoninic acid method. Bivariate and multivariate (generalized linear modeling) analyses evaluated the relationships between age, gender and salivary protein concentrations. Results: Mean salivary levels of SLPI and lysozyme were lower in elderly compared with non-elderly subjects (p < 0.001), unlike lactoferrin and total protein levels. Similar results were obtained when concentrations of the individual proteins were normalized to the total protein concentration, suggesting that glandular production of SLPI and lysozyme preferentially decreases with aging. Gender differences were detected only for SLPI concentrations; males had lower SLPI levels than females regardless of age (p < 0.01). Generalized linear models confirmed that age (p < 0.001) and gender (p < 0.05) were each associated with the SLPI concentration and together accounted for 50% of the variation in SLPI concentration in this population. Conclusion: These findings indicate that SLPI production is diminished among healthy community-dwelling older adults, particularly elderly males. Further investigation should determine the impact of decreased local SLPI production on the increased risk of oral mucosal disease with advanced age.

Production of secretory leukocyte protease inhibitor by human amniotic membranes and regulation of its concentration in amniotic fluid.

Secretory leukocyte protease inhibitor (SLPI) is a potent inhibitor of human leukocyte elastase. SLPI is a protein found in various human fluids, including parotid secretions, cervical mucus, seminal plasma and ascites. Western blot analysis revealed that SLPI protein is detected as a 12 kDa band in both the amniotic fluid and the amniotic membrane. The amniotic fluid concentrations of SLPI were assayed by enzyme-linked immunosorbent assay. SLPI concentrations in the amniotic fluid of women in the third trimester were higher than those in the second trimester. Immunohistochemistry using an anti-SLPI polyclonal antibody revealed positive staining in epithelial cells in amniotic membranes. Reverse transcription-polymerase chain reaction demonstrated that SLPI transcripts could be detected in the amniotic membranes. To determine the mechanism of SLPI production by amniotic cells, purified amniotic cells were stimulated with various cytokines. Amniotic cells produced SLPI in a dose-dependent manner when stimulated with interleukin (IL)-1alpha, IL-1beta, and tumour necrosis factor-alpha. The present findings show that SLPI is produced by the amniotic membranes in response to cytokine concentrations. The SLPI in the amniotic fluid may contribute to immunodefence mechanisms during pregnancy.

The presence of elafin, SLPI, IL1-RA and STNFalpha RI in head and neck squamous cell carcinomas and their relation to the degree of tumour differentiation.

Biopsy samples of head and neck carcinomas were investigated with regard to elafin, secretory leukocyte protease inhibitor (SLPI), interleukin 1-receptor antagonist [(IL)1-RA] and soluble tumour necrosis factor alpha receptor antagonist (STNFalpha RI). SLPI and elafin are serine protease inhibitors produced in the serous cells of the upper respiratory airways and in the keratinocytes, respectively. We have now found the presence of elafin and SLPI in squamous cell carcinomas of the upper respiratory tract (tonsillar, hypopharyngeal, tongue, mouth floor, gingival and laryngeal cancer). Significantly higher amounts of SLPI and elafin are present in well-differentiated and moderately differentiated tumours than in poorly differentiated tumours (p < 0.0001 and p < 0.0015). Tumour necrosis factor-alpha and IL-1beta have been shown to stimulate the production of SLPI and elafin. Since these cytokines can both be difficult to detect, we chose to study their inhibitors, STNFalpha RI and IL1-RA, instead. IL1-RA was expressed in highly differentiated tumours as well as in poorly differentiated ones. No significant difference was seen between the groups. STNFalpha RI was only found in very small amounts, sparsely distributed in the tumours, and was not related to the degree of differentiation.

Secretory leukocyte protease inhibitor (SLPI) concentrations in cervical mucus of women with normal menstrual cycle.

Secretory leukocyte protease inhibitor (SLPI) is a potent inhibitor of human leukocyte elastase. SLPI transcripts in the cervical tissue were detected during the menstrual cycle by reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis revealed that the intensity of SLPI protein in cervical tissue in the ovulatory phase was stronger than in other phases. Immunohistochemistry using an anti-SLPI polyclonal antibody revealed positive staining in the epithelial cells of the endocervix. Western blot analysis also revealed that SLPI protein was present in the cervical mucus. Again the intensity of SLPI protein in the ovulatory phase was stronger than that in the follicular phase. The SLPI concentrations and SLPI/elastase ratios in the cervical mucus of women in the ovulatory phase were significantly higher than in the follicular and luteal phases. The SLPI and elastase concentrations in the cervical mucus were positively correlated. No significant difference was found in the SLPI serum concentrations of women during the menstrual cycle. These results suggest that production of SLPI from cervical epithelial cells during the ovulatory phase may be important for protection from the effects of elastase.

Anti-inflammatory function of secretory leukocyte protease inhibitor

Macrophages respond to lipopolysaccharide (LPS) by producing a battery of alarm signals and defense molecules, a response that can be beneficial or detrimental to the host depending on its scale. To explore the regulation of LPS response, we compared cell lines from two strains of mice congenic for a locus markedly affecting LPS sensitivity. Differential display detected a transcript expressed in a macrophage cell line derived from LPS-hyporesponsive mice ( Lpsd) but not in macrophages derived from congenic LPS-responsive mice ( Lps n). The cloned cDNA encodes a protein homologous to human secretory leukocyte protease inhibitor (SLPI), previously regarded as an epithelial cell-derived inhibitor of leukocyte serine proteases. In macrophages, SLPI production correlates inversely with response to LPS. Phagocytes are important sources of SLPI. Besides LPS, taxol, lipoteichoic acid (LTA) and two anti-inflammatory cytokines, IL-10 and IL-6, are capable of inducing SLPI expression in macrophages. Stable transfection with SLPI suppresses the LPS- and LTA-induced production of nitric oxide and TNFα by macrophages. An anti-inflammatory role for macrophage-derived SLPI seems likely based on the following: (i) the slowly increasing production of SLPI in response to constituents of Gram-negative and Gram-positive bacteria; (ii) its induction both as a direct response to LPS and as a response to anti-inflammatory cytokines (IL-6 and IL-10) induced by LPS; and (iii) its ability to suppress the production of pro-inflammatory products by macrophages stimulated with constituents of both Gram-positive and Gram-negative bacteria.

Corticosteroids increase secretory leukocyte protease inhibitor transcript levels in airway epithelial cells.

Secretory leukocyte protease inhibitor (SLPI) is the predominant antiprotease of the conducting airways and may play a role in reducing airway inflammation. In this study, the effect of corticosteroids used in the treatment of inflammatory airway disease on SLPI transcript levels was investigated. When human airway epithelial cells (9HTEo-) were treated continuously with 10 nM fluticasone propionate, SLPI transcript levels increased within 12 h, with maximal transcript accumulation occurring at 24-48 h. Several corticosteroids (0.1-1,000 nM) were compared, and the following potency in increasing SLPI transcript levels was observed: fluticasone > triamcinolone > or = dexamethasone > methylprednisolone > hydrocortisone. Fluticasone, the most potent corticosteroid, increased SLPI transcript levels at doses as low as 0.1 nM, whereas hydrocortisone, the least potent corticosteroid, was effective at 100 nM. Fluticasone-induced increases in SLPI transcript levels were inhibited by cycloheximide, suggesting protein synthesis may be required for this response. Because proteases are likely to be present when corticosteroids are administered therapeutically, we examined the interaction between elastase and fluticasone and found they act synergistically to increase SLPI transcript levels. Our findings suggest that corticosteroids may exert their antiinflammatory effects in part by increasing airway epithelial cell SLPI production.