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.     

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.     

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.     

Cleaved SLPI, a novel biomarker of chymase activity

Secretory leukocyte protease inhibitor (SLPI) is a protease inhibitor of the whey acidic protein-like family inhibiting chymase, chymotrypsin, elastase, proteinase 3, cathepsin G and tryptase. Performing in vitro enzymatic assays using both Western blotting and liquid chromatography/mass spectrometry techniques we showed that, of the proteases known to interact with SLPI, only chymase could uniquely cleave this protein. The peptides of the cleaved SLPI (cSLPI) remain coupled due to the disulfide bonds in the molecule but under reducing conditions the cleavage can be observed as peptide products. Subsequent ex vivo studies confirmed the presence of SLPI in human saliva and its susceptibility to cleavage by chymase. Furthermore, inhibitors of chymase activity are able to inhibit this cleavage. Human saliva from both normal and allergic individuals was analyzed for levels of cSLPI and a correlation between the level of cSLPI and the extent of allergic symptoms was observed, suggesting the application of cSLPI as a biomarker of chymase activity in humans.     

Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor with anti-microbial properties found in mucosal fluids. It is expressed during cutaneous wound healing. Impaired healing states are characterized by excessive proteolysis and often bacterial infection, leading to the hypothesis that SLPI may have a role in this process. We have generated mice null for the gene encoding SLPI (Slpi), which show impaired cutaneous wound healing with increased inflammation and elastase activity. The altered inflammatory profile involves enhanced activation of local TGF-beta in Slpi-null mice. We propose that SLPI is a pivotal endogenous factor necessary for optimal wound healing.     

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.     

Helicobacter pylori-induced downregulation of the secretory leukocyte protease inhibitor (SLPI) in gastric epithelial cell lines and its functional relevance for H. pylori-mediated diseases

The secretory leukocyte protease inhibitor (SLPI) exerts antiproteolytic activity towards serine proteases, as well as anti-microbial and anti-inflammatory effects. To investigate its role in H. pylori-mediated diseases, SLPI expression was analyzed by RT-PCR, ELISA and immunohistochemistry in clinical samples and gastric tumor cell lines. Determination of the mucosal SLPI levels in 126 patients confirmed the previously reported downregulation of SLPI in H. pylori-infected patients. The lower SLPI levels in antral biopsies of H. pylori-positive subjects were associated with a 30-fold increase (p<0.01) in neutrophil elastase activity, and a significant negative correlation was demonstrated for both parameters (R=-0.63, p=0.0002). Eradication of the bacterium in a long-term study (5-7 years) led to a recovery of mucosal SLPI expression. In vitro experiments using four gastric tumor cell lines (AGS, MKN-28, MKN-45, NCI-N87) generally confirmed the clinical findings. While the co-incubation of these cell lines with H. pylori resulted in lower or unchanged SLPI protein levels, the corresponding SLPI mRNA amounts were upregulated by up to five-fold (p=0.006) in all cell lines. Taken together, these results indicate that the reduction in antral SLPI levels in H. pylori-infected subjects has a functional relevance for gastric mucosa and the H. pylori-induced decrease in SLPI is primarily regulated at the posttranslational level.     

Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair

Increased leukocyte elastase activity in mice lacking secretory leukocyte protease inhibitor (SLPI) leads to impaired wound healing due to enhanced activity of TGFbeta and perhaps additional mechanisms. Proepithelin (PEPI), an epithelial growth factor, can be converted to epithelins (EPIs) in vivo by unknown mechanisms with unknown consequences. We found that PEPI and EPIs exert opposing activities. EPIs inhibit the growth of epithelial cells but induce them to secrete the neutrophil attractant IL-8, while PEPI blocks neutrophil activation by tumor necrosis factor, preventing release of oxidants and proteases. SLPI and PEPI form complexes, preventing elastase from converting PEPI to EPIs. Supplying PEPI corrects the wound-healing defect in SLPI null mice. Thus, SLPI/elastase act via PEPI/EPIs to operate a switch at the interface between innate immunity and wound healing.     

Isolation of chymase complexed with physiological inhibitor similar to secretory leukocyte protease inhibitor (SLPI) from hamster cheek pouch tissues

A low molecular weight protein complexed with chymase was isolated from hamster cheek pouch tissues. This protein had an apparent molecular mass of about 10 kDa on SDS-PAGE and the N-terminal sequence showed some homology to secretory leukocyte protease inhibitor (SLPI), which is known as the predominant inhibitor of neutrophil elastase and cathepsin G. Remarkably enhanced inhibition of chymase activity was achieved in the presence of heparin, indicating that the functional property was also similar to SLPI. These findings suggest that this SLPI-like protein is a candidate for a physiological inhibitor of chymase.     

Localization of a low molecular weight protease inhibitor to tracheal and mixillary sinus mucosa.

Normal human bronchial secretion contains protease inhibitors from plasma and an acid-stable, low molecular weight inhibitor which strongly inactivates granulocyte elastase and chymotrypsin-like cationic proteins. The distribution of this bronchial protease inhibitor in tracheal mucosa was analyzed by an indirect immunoperoxidase method. Strong peroxidase staining was obtained in the columnar ciliated epithelium of the trachea and in the sero-mucus glands. The findings support a local mucosal production of the inhibitor. Maxillary sinus mucosa was also positively stained for the inhibitor.     

Development of affinity chromatography using a bioactive peptide as a ligand

By repeatedly introducing hydrophilic polyethylene glycol (PEG) spacer (2) onto affinity resin bearing a bioactive peptide (1/2 secretory leukocyte protease inhibitor, 1/2SLPI) as a ligand, the adsorption of nonspecific binding proteins was effectively reduced and the purification efficacy of elastase, which is one of the target molecules for 1/2SLPI, from a protein mixture was improved. Moreover, using this resin, we also successfully detected L-plastin, as an endogenous target molecule for SLPI, from HL-60 cell lysate.     

Cathepsin B, L, and S cleave and inactivate secretory leucoprotease inhibitor.

A number of serine proteases, matrix metalloproteases, and cysteine proteases were evaluated for their ability to cleave and inactivate the antiprotease, secretory leucoprotease inhibitor (SLPI). None of the serine proteases or the matrix metalloproteases examined cleaved the SLPI protein. However, incubation with cathepsins B, L, and S resulted in the cleavage and inactivation of SLPI. All three cathepsins initially cleaved SLPI between residues Thr(67) and Tyr(68). The proteolytic cleavage of SLPI by all three cathepsins resulted in the loss of the active site of SLPI and the inactivation of SLPI anti-neutrophil elastase capacity. Cleavage and inactivation were catalytic with respect to the cathepsins, so that the majority of a 400-fold excess of SLPI was inactivated within 15 min by cathepsins L and S. Analysis of epithelial lining fluid samples from individuals with emphysema indicated the presence of cleaved SLPI in these samples whereas only intact SLPI was observed in control epithelial lining fluid samples. Active cathepsin L was shown to be present in emphysema epithelial lining fluid and inhibition of this protease prevented the cleavage of recombinant SLPI added to emphysema epithelial lining fluid. Taken together with previous data that demonstrates that cathepsin L inactivates alpha(1)-antitrypsin, these findings indicate the involvement of cathepsins in the diminution of the lung antiprotease screen possibly leading to lung destruction in emphysema.     

Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family

Elafin and SLPI are low-molecular weight proteins that were first identified as protease inhibitors in mucous fluids including lung secretions, where they help control excessive proteolysis due to neutrophil serine proteases (elastase, proteinase 3 and cathepsin G). Elafin and SLPI are structurally related in that both have a fold with a four-disulfide core or whey acidic protein (WAP) domain responsible for inhibiting proteases. Elafin is derived from a precursor, trappin-2 or pre-elafin, by proteolysis. Trappin-2, which is itself a protease inhibitor, has a unique N-terminal domain that enables it to become cross-linked to extracellular matrix proteins by transglutaminase(s). SLPI and elafin/trappin-2 are attractive candidates as therapeutic molecules for inhibiting neutrophil serine proteases in inflammatory lung diseases. Hence, they have become the WAP proteins most studied over the last decade. This review focuses on recent findings revealing that SLPI and elafin/trappin-2 have many biological functions as diverse as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory and immuno-modulatory functions, in addition to their well-recognized role as protease inhibitors.     

Quantitative Proteomic Analysis of Oral Brush Biopsies Identifies Secretory Leukocyte Protease Inhibitor as a Promising,Mechanism-Based Oral Cancer Biomarker

A decrease in the almost fifty percent mortality rate from oral cancer is needed urgently. Improvements in early diagnosis and more effective preventive treatments could affect such a decrease. Towards this end, we undertook for the first time an in-depth mass spectrometry-based quantitative shotgun proteomics study of non-invasively collected oral brush biopsies. Proteins isolated from brush biopsies from healthy normal tissue, oral premalignant lesion tissue (OPMLs), oral squamous cell carcinoma (OSCC) and matched control tissue were compared. In replicated proteomic datasets, the secretory leukocyte protease inhibitor (SLPI) protein stood out based on its decrease in abundance in both OPML and OSCC lesion tissues compared to healthy normal tissue. Western blotting in additional brushed biopsy samples confirmed a trend of gradual decreasing SLPI abundance between healthy normal and OPML tissue, with a larger decrease in OSCC lesion tissue. A similar SLPI decrease was observed in-vitro comparing model OPML and OSCC cell lines. In addition, exfoliated oral cells in patients’ whole saliva showed a loss of SLPI correlated with oral cancer progression. These results, combined with proteomics data indicating a decrease in SLPI in matched healthy control tissue from OSCC patients compared to tissue from healthy normal tissue, suggested a systemic decrease of SLPI in oral cells correlated with oral cancer development. Finally, in-vitro experiments showed that treatment with SLPI significantly decreased NF-kB activity in an OPML cell line. The findings indicate anti-inflammatory activity in OPML, supporting a mechanistic role of SLPI in OSCC progression and suggesting its potential for preventative treatment of at-risk oral lesions. Collectively, our results show for the first time the potential for SLPI as a mechanism-based, non-invasive biomarker of oral cancer progression with potential in preventive treatment.     

分泌性白细胞蛋白酶抑制因子在肿瘤中的作用

分泌性白细胞蛋白酶抑制因子(secretory leukocyte protease inhibitor, SLPI)是一个可抑制多种丝氨酸蛋白酶活性的阳离子蛋白质。SLPI羧基端具有抑制糜蛋白酶、胰弹性蛋白酶等抗蛋白酶活性，氨基端的功能尚不清楚，可能具有抗菌、抗真菌、抗病毒、抗炎和免疫调节等活性。近年来研究发现，SLPI在有些癌症，如卵巢癌、肺癌、胃癌、结肠癌中表达升高，但在有些癌症如乳腺癌、前列腺癌、口腔癌中表达降低。目前，尚未完全了解SLPI在调控致癌效应中的作用。本文就SLPI在肿瘤及抗肿瘤中的可能作用及其机制进行综述，为SLPI在抗肿瘤中的应用提供新思路。     

Secretory leukocyte protease inhibitor (SLPI) is, like its homologue trappin-2 (pre-elafin), a transglutaminase substrate

Human lungs contain secretory leukocyte protease inhibitor (SLPI), elafin and its biologically active precursor trappin-2 (pre-elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases.     

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.     

A protease inhibitor produced by Streptomyces lividans 66 exhibits inhibitory activities toward both subtilisin BPN' and trypsin.

A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.