Characterization of secretory leukocyte protease inhibitor as an inhibitor of implantation serine proteinases

We have recently identified and characterized two implantation serine proteinase genes, ISP1 and ISP2, which give rise to a dimeric proteinase, ISP that facilitates embryo invasion during peri-implantation period. As many proteinases have cognate serpins that regulate their proteolytic activity, we have been investigating anti-tryptases, expressed during this window of implantation. Here, we report the differential expression of secretory leukocyte protease inhibitor (SLPI) in uterine endometrium around the implantation period. The co-localization of SLPI and ISP suggests the possibility that SLPI is an ISP serpin and that expression of SLPI may lead to a reduction in ISP activity. The expression of SLPI is down regulated during the window of embryo-uterine receptivity. Our results are consistent with a model suggesting that the drop in SLPI expression may help to refine the opening of the window of implantation, by allowing the proteolytic activity of embryo invasive serine proteinases such as the ISPs.     

Estrogen induces expression of secretory leukocyte protease inhibitor in rat uterus

In rodents, the steroid hormone estrogen (E) profoundly influences the early events in the uterus leading to embryo implantation. It is thought that E triggers the expression of a unique set of genes in the endometrium that in turn control implantation. To identify these E-induced genes, we employed a delayed implantation model system in which embryo attachment to rat endometrium is dependent upon E administration. Using a gene expression screen method, we isolated a number of cDNAs representing mRNAs whose expression is either turned on or turned off in response to an implantation-inducing dose of E. We identified one of these cDNAs as that encoding secretory leukocyte protease inhibitor (SLPI), an inhibitor of serine proteases. The expression of SLPI mRNA was induced in the uteri of ovariectomized rats in response to E, confirming the hormonal regulation of this molecule. Spatiotemporal analysis revealed a biphasic pattern of expression of SLPI mRNA during early pregnancy. A considerable amount of SLPI mRNA was detected in the uterine epithelium on Day 1 of pregnancy. The level of this mRNA, however, declined sharply on Days 2 and 3 of gestation. Interestingly, on Day 4 of gestation, there was a marked resurgence in SLPI mRNA expression in the uterine epithelium. This second burst of SLPI expression diminished by Day 6 of pregnancy. The transient induction of SLPI mRNA during Days 4 and 5 overlapped with the window of implantation in the rat. Although the precise function of SLPI in the uterus eludes us presently, its known effects as a serine protease inhibitor in other tissues and its hormone-induced expression in the rat uterus immediately preceding implantation lead us to propose that this gene plays an important role in controlling excessive proteolysis and inflammation during a critical phase of early pregnancy.     

The tumor-promoting effect of TNF-alpha involves the induction of secretory leukocyte protease inhibitor

According to the cancer immunoediting concept, inflammatory mediators play not only a critical role in promoting host protection against cancer but also contribute to cancer cell growth and survival. TNF-alpha is a critical factor in this network. However, the mechanisms underlying the tumor-promoting effect of TNF-alpha have not been fully elucidated yet. We previously reported that in vitro culture of Lewis lung carcinoma 3LL cells with TNF-alpha-producing macrophages resulted in enhanced resistance toward TNF-alpha-mediated lysis and increased malignancy of the 3LL cells. In this study, we analyzed the effects of endogenous TNF-alpha on TNF-alpha resistance and malignant behavior in vivo of low-malignant/TNF-alpha-sensitive 3LL-S cells and cancer cells derived from 3LL-S tumors that developed in wild-type or TNF-alpha(-/-) mice. Interestingly, 3LL-S cells acquired a malignant phenotype in vivo depending on the presence of host TNF-alpha, whereas acquisition of TNF-alpha resistance was TNF-alpha-independent. This result suggested that malignancy-promoting characteristics of 3LL-S cells other than TNF-alpha resistance are influenced in vivo by TNF-alpha. We previously identified the malignancy-promoting genes, secretory leukocyte protease inhibitor (SLPI) and S100A4, as being up-regulated in 3LL-S cells upon their s.c. growth in wild-type mice. In this study, we show that SLPI, but not S100A4, was induced in 3LL-S cells both in vitro and in vivo by TNF-alpha, and that silencing of in vivo induced 3LL-S SLPI expression using RNA interference abrogated in vivo progression but did not influence TNF-alpha resistance. These data indicate that SLPI induction may be one mechanism whereby TNF-alpha acts as an endogenous tumor promoter.     

Renaturation of recombinant secretory leukocyte protease inhibitor: Aspects of disulfide bond formation kinetics

Summary Therapeutic proteins produced in procaryotic hosts often contain disulfide bonds, which must be fully formed to satisfy United States Food and Drug Administration regulations. Native secretory leukocyte protease inhibitor (SLPI), a possible emphysema therapeutic agent, contains many disulfide bonds. However, when SLPI is produced in Escherichia coli by rDNA technology, the disulfide bonds are not formed correctly and must be generated by in vitro renaturation. In this study, the reaction rate parameters were estimated for SLPI renaturation. The apparent activation energy was approximately 5 kcal/mol suggesting that renaturation is a diffusion limited process. Apparent reaction rate orders were not constant, suggesting complex renaturation mechanism(s).Currently at Texas A & M University, College Station, Texas 77843, Department of Chemical Engineering.     

Role of secretory protease inhibitor SPINK3 in mouse uterus during early pregnancy

Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.     

Construction,non-denaturing affinity purification,and characterization of baculovirally expressed human secretory leukocyte protease inhibitor

Secretory leukocyte protease inhibitor (SLPI) is a 11.7 kDa mucosal protein with potent anti-microbial, anti-inflammatory, and wound healing activities. Previous efforts to express and purify the non-glycosylated cationic protein as a recombinant protein in bacteria required extensive denaturation and renaturation to refold the disulfide-rich protein into its biologically active form. To overcome this limitation, we have expressed human SLPI as a polyhistidine-tagged protein (bvHisSLPI) using a recombinant baculovirus expression system. Studies were conducted to determine the timing of maximal protein production following baculovirus infection of Sf21 cells. The 16.4kDa-tagged protein was then overexpressed in Sf21 cells following a 48-h infection with bvHisSLPI-encoding baculovirus, purified by nickel-chelating affinity chromatography under non-denaturing conditions, and analyzed by Coomassie-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Purified bvHisSLPI was further characterized by enterokinase digestion to remove the polyhistidine tag from its N-terminus. In serine protease inhibition assays, purified bvHisSLPI blocked substrate cleavage by two serine proteases, chymotrypsin and cathepsin G, comparable to bacterially expressed SLPI. The baculovirus expression and affinity purification strategy described here will facilitate further studies of the structural and biological properties of this important multifunctional protein.     

Suppression of macrophage responses to bacterial lipopolysaccharide by a non-secretory form of secretory leukocyte protease inhibitor.

Expression of secretory leukocyte protease inhibitor (SLPI) suppresses the ability of macrophages to respond to bacterial lipopolysaccharide (LPS). Here, addition of recombinant or native SLPI to the extracellular medium was non-suppressive, while transfection with a non-secretory form of SLPI was fully suppressive, an effect overcome by treatment with interferon-gamma. A portion of the SLPI produced by untransfected macrophages was localized in the cytosol. Thus, SLPI can act intracellularly to block macrophage activation by LPS.     

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.     

Effects of secretory leukocyte protease inhibitor on the tumor necrosis factor-alpha production and NF-kappaB activation of lipopolysaccharide-stimulated macrophages

It has been reported that lipopolysaccharide (LPS)-hyporesponsiveness of macrophages (Mphis) of C3H/HeJ mice with a mutated Lps gene (Lps(d)) is related to high-level expression of secretory leukocyte protease inhibitor (SLPI) in response to LPS, causing suppression of NF-kappaB activation and tumor necrosis factor-alpha (TNF-alpha) production. We thus examined the effects of SLPI on the TNF-alpha production by LPS-stimulated Mphis. Neither intact SLPI nor half-sized SLPI (1/2 SLPI) down-regulated Mphi TNF-alpha production. 1/2 SLPI weakly increased Mphi TNF-alpha production in response to LPS signaling and potentiated the LPS-induced activation of NF-kappaB, especially the binding of p65-p50 heterodimers to the DNA kappaB sites, suggesting that LPS-hyporesponsiveness of Lps(d) Mphis is not due to the overexpression of SLPI.     

Trichuris suis: A secretory serine protease inhibitor

A trypsin inhibitor was identified in extracts of adult Trichuris suis and culture fluids from 24-h in vitro cultivation of adult parasites. The inhibitor was isolated by acid precipitation, affinity chromatography (trypsin-agarose), and reverse phase HPLC as a single polypeptide with a molecular weight estimated at 6.6 kDa by laser desorption mass spectrometry. The purified inhibitor associated strongly with trypsin (equilibrium dissociation inhibitory constant (K(j)) of 3.07 nM) and chymotrypsin (K(j) = 24.5 nM) and was termed TsTCI. Elastase, thrombin, and factor Xa were not inhibited. The cDNA-derived amino acid sequence of the mature TsTCI consisted of 61 residues including 8 cysteine residues with a molecular mass of 6.687 kDa. The N-terminal region of TsTCI (46 residues) showed limited homology (36%) to a protease inhibitor from the hemolymph of the honeybee Apis mellifera, which is considered to be a member of the Ascaris inhibitor family. However, TsTCI did not display sequence homology with other members of this family or the distinctive cysteine residue pattern which distinguishes this family. However, similarity of a region of TsTCI (11 residues) with the reactive site regions of inhibitors from the nematodes Ascaris suum, Anisakis simplex, and Ancylostoma caninum was apparent.     

Expression and characterization of recombinant human secretory leukocyte protease inhibitor (SLPI) protein from Pichia pastoris

The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7 kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.     

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.     

Secretory leukocyte protease inhibitor is a novel inhibitor of fibroblast-mediated collagen gel contraction

Cultured epithelial cells, including those from the oral epithelium, have been successfully applied in the promotion of scarless wound healing. Factors released from the epithelial cells are thought to contribute significantly to the beneficial effects. In the conditioned medium of human oral epithelial cells, we found a factor that inhibited fibroblast-mediated collagen gel contraction, an in vitro model of wound healing and scar formation. Biochemical analysis identified the factor to be human secretory leukocyte protease inhibitor (SLPI). Fibroblasts transfected with SLPI cDNA showed reduced gel-contracting activity. SLPI purified from the conditioned medium inhibited gel contraction in a dose-dependent manner, and anti-SLPI antibody counteracted this activity. Upon SLPI treatment, human skin fibroblasts in collagen gel became shorter in length and were inhibited in pseudopodia extension. Furthermore, after SLPI treatment, alpha(1)-integrin immunoreactivity decreased, and cyclic AMP levels increased. Excessive gel contraction was observed when fibroblasts treated with TGF-beta1 and fibroblasts from hypertrophic and from keloid scar tissue were cultured in collagen gel. SLPI was also effective in inhibiting gel contraction in the above three models of scar formation. These results suggest that SLPI may be useful in promoting scarless wound healing.     

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.