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.     

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.     

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.     

The phenotype of inflammatory macrophages is stimulus dependent: implications for the nature of the inflammatory response

Many diseases are characterized by inflammatory reactions involving both the innate and adaptive arms of the immune system. Thioglycolate medium (TM) injection into the peritoneal cavity has long been used as a stimulus for eliciting inflammatory macrophages for study and for determining the importance of a particular mediator in inflammation. However, the response to this irritant may not be relevant to many inflammatory diseases. Therefore, we have developed an Ag-specific peritonitis model using methylated BSA (mBSA) as the stimulus. Priming mice intradermally with mBSA in adjuvant and boosting 14 days later, followed by an i.p. challenge with mBSA after an additional 7 days, led to an inflammatory reaction equivalent in magnitude to that induced with TM as judged by the number of exudate cells. The inflammatory macrophages elicited by the mBSA protocol differed, being smaller and less vacuolated than TM-elicited macrophages. Also, macrophages from 4-day mBSA-induced exudates expressed more MHC class II than TM-induced exudates, were able to stimulate allogeneic T lymphocytes, and upon in vitro stimulation with LPS secreted greater levels of IL-6 and IL-1beta. Macrophages from 4-day TM-induced exudates, on the other hand, expressed Ly6C and ER-MP58, immature myeloid markers. The inflammatory response elicited using the Ag mBSA may be more relevant for studying the inflammatory responses in many diseases, such as those of autoimmune origin and those involving an acquired immune response.     

CD14-dependent clearance of apoptotic cells by human macrophages: the role of phosphatidylserine

Apoptotic-cell clearance is dependent on several macrophage surface molecules, including CD14. Phosphatidylserine (PS) becomes externalised during apoptosis and participates in the clearance process through its ability to bind to a novel receptor, PS-R. CD14 has the proven ability to bind phospholipids and may function as an alternative receptor for the externalised PS of apoptotic cells. Here we demonstrate that CD14 does not function preferentially as a PS receptor in apoptotic-cell clearance. Compared with phosphatidylcholine and phosphatidylethanolamine, PS was the least active phospholipid binding to human monocyte-derived macrophages and showed no specificity for soluble or membrane-anchored CD14. Significantly, PS-containing liposomes failed to inhibit CD14-dependent uptake of apoptotic cells by macrophages. PS exposure was, however, found to be insufficient for either CD14-dependent or CD14-independent apoptotic-cell uptake by phagocytes. The additional features that enable apoptotic-cell clearance are derived from mechanisms that can be divorced temporally from those responsible for the morphological features of apoptosis.     

Chlamydia trachomatis and Chlamydophila abortus induce the expression of secretory leukocyte protease inhibitor in cells of the human female reproductive tract

C. trachomatis and C. abortus are related Gram‐negative intracellular bacteria that cause reproductive failure due to infertility (C. trachomatis) or abortion (C. abortus). These organisms target epithelial cells in the reproductive tract and/or placenta, but the innate immune mechanisms that lead to protection or pathology and disease are poorly understood. SLPI is an innate immune molecule which protects mucosal surfaces from infection and injury. C. trachomatis and C. abortus were found to induce SLPI mRNA and peptide expression in HeLa (cervical epithelium) and JEG‐3 cells (trophoblast) respectively. Both cell lines constitutively expressed SLPI and, although infection enhanced this expression, killed organisms did not. These data demonstrate that Chlamydia/Chlamydophila grow in cells that express SLPI, suggesting that SLPI does not exert antimicrobial effects against these organisms. However, SLPI has multiple functions, and we speculate that it may play a role in controlling tissue inflammation and pathology.     

Autophagy gene-dependent clearance of apoptotic cells during embryonic development

Autophagy is commonly observed in metazoan organisms during programmed cell death (PCD), but its function in dying cells has been unclear. We studied the role of autophagy in embryonic cavitation, the earliest PCD process in mammalian development. Embryoid bodies (EBs) derived from cells lacking the autophagy genes, atg5 or beclin 1, fail to cavitate. This defect is due to persistence of cell corpses, rather than impairment of PCD. Dying cells in autophagy gene null EBs fail to express the "eat-me" signal, phosphatidylserine exposure, and secrete lower levels of the "come-get-me" signal, lysophosphatidylcholine. These defects are associated with low levels of cellular ATP and are reversed by treatment with the metabolic substrate, methylpyruvate. Moreover, mice lacking atg5 display a defect in apoptotic corpse engulfment during embryonic development. We conclude that autophagy contributes to dead-cell clearance during PCD by a mechanism that likely involves the generation of energy-dependent engulfment signals.     

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.     

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.