Secretory Leukocyte Protease Inhibitor (SLPI)
Secretory leukocyte protease inhibitor (SLPI) was first described by Robert Thompson and Kjell Ohlsson in a 1986 study that aimed to identify novel leukocyte protease inhibitors in human saliva (Thompson and Ohlsson 1986). In their experiments, SLPI was purified from human parotid saliva using Sephadex columns, and the protein was found to have a molecular weight of 11.7 kD. Subsequent studies determined that high levels of SLPI are also present in bronchial mucus, seminal fluid, lacrimal secretions, breast milk, and cervical mucus. The biochemical analyses conducted by Thompson and Ohlsson revealed that SLPI inhibited the activity of leukocyte (neutrophil) elastase, cathepsin G, trypsin, and chymotrypsin. These findings established SLPI as a potent and broad-spectrum inhibitor of serine proteases, and it is functionally related to other antiproteases such as α1-antitrypsin and elafin. Since its discovery, SLPI has been characterized as an alarm antiprotease that protects the oral, bronchial, and urogenital mucosae from inflammation and proteolytic damage.
Human SLPI is synthesized as a single polypeptide chain measuring 132 amino acids in length. A N-terminal 25-amino acid signal peptide is cleaved prior to secretion of the protein, resulting in a mature, non-glycosylated protein composed of 107 amino acids. One of the most unique aspects of SLPI is its amino acid composition, as four amino acids account for nearly 50% of the polypeptide sequence. SLPI contains 16 cysteine residues (15%), extremely high levels of proline (13 residues – 12%), and a high percentage of lysine and arginine (16%) that contributes to the cationic nature of the protein (Grütter et al. 1988). The tertiary structure of SLPI resembles that of a boomerang, with a short linking region joining the N-terminal domain to the C-terminal domain (Grütter et al. 1988). These domains have a high degree of homology and stability, with each domain displaying a planar structure that consists of a β-hairpin loop surrounded by a wider outer loop (Grütter et al. 1988). The domain cores are notable for their lack of hydrophobicity. The polypeptide chains of each domain are covalently linked by four highly conserved disulfide bonds, forming a structure known as a whey acidic protein four-disulfide core (WFDC) motif. SLPI is therefore commonly classified as a member of the WFDC family of proteins and is occasionally referred to as WFDC4. The WFDC protein family has over 20 members, including anosmin-1 (WFDC19), epididymal peptidase inhibitor (WFDC7), peptidase inhibitor 3 (WFDC14), and semenogelins-1 and semenogelins-2. The majority of these proteins are encoded at a single locus (WFDC locus) on chromosome 20 in humans, but the genes encoding other WFDC-containing proteins such as WAP, follistatin/Kazal, immunoglobulin, Kunitz and netrin domain containing 1 (WFIKKN1) are found at other sites within the genome. Due to the highly conserved nature of WFDC domains, there is a high degree of sequence identity in mammalian SLPI. Human SLPI shares 60% amino acid sequence identity with mouse SLPI and 63% identity with rat SLPI (Wang et al. 2003). SLPI-like proteins have also been identified in invertebrates such as the whiteleg shrimp (Litopenaeus vannamei; Jiménez-Vega and Vargas-Albores 2007) and human whipworm (Trichuris trichiura; Foth et al. 2014).
SLPI and Microorganisms
SLPI also displays antiviral properties, and perhaps its best-known function is its ability to inhibit infection of monocytes and macrophages by human immunodeficiency virus (HIV). It had been known since the mid-1980s that oral transmission of HIV was extremely low and that human saliva possessed significant anti-HIV activity that was not present in other bodily fluids such as plasma, urine, synovial fluid, and cerebrospinal fluid (McNeely et al. 1995). This prompted several studies aimed at identifying the factors in human saliva that were responsible for this effect. In an in vitro study that examined HIV infection of monocytes and T lymphocytes, SLPI was the only salivary protein that displayed significant anti-HIV activity at physiological concentrations (McNeely et al. 1995). Other salivary proteins such as statherin, cystatin, lactoferrin, and lysozyme failed to inhibit infection or only inhibited infection at nonphysiological concentrations. Depletion of SLPI from saliva led to a 50% reduction in its ability to prevent HIV infection, indicating that SLPI is primarily responsible for the anti-HIV activity in saliva.
SLPI does not directly affect the survival or function of HIV itself, but rather mediates its effects by blocking the interaction of the virus with proteins on the surfaces of target cells. It was first shown that SLPI can bind to annexin II on macrophages and that the presence of SLPI disrupts the interaction of annexin II with the human phosphatidylserine that is acquired by HIV from host cells and displayed on the outer coat of the virus (Ma et al. 2004). It was proposed that disrupting the interaction with annexin II does not prevent binding of HIV to the target cell, but rather inhibits postbinding and the pre-reverse transcription phase of the viral life cycle. In 2009, phospholipid scramblase 1 (PLSCR1) was identified as a receptor for SLPI on the surfaces of T lymphocytes (Py et al. 2009). The cytoplasmic domain of PLSCR1 interacts with CD4, the receptor that binds HIV on T lymphocytes, and it has been suggested that the interaction between PLSCR1 and CD4 facilitates fusion of HIV with the plasma membrane of the host cell. It was shown that SLPI can disrupt this interaction by competitively binding to the same site on PLSCR1, and it was therefore proposed that SLPI may inhibit HIV infection of T lymphocytes by modulating CD4 function. To date, there has been no further insight into the mechanism that underlies SLPI’s ability to inhibit HIV infection, but this remains an active area of study within the field.
SLPI and Inflammation
SLPI plays an important role in suppressing the inflammatory responses that occur in response to tissue injury. In mice with a germ line null mutation of SLPI (Slpi –/– ), cutaneous wound healing was severely impaired, and there was increased inflammation at the site of injury that was associated with extensive infiltration of neutrophils and macrophages (Ashcroft et al. 2000). Elastase activity was also elevated within the lesion site, and this led to the assertion that the collagen matrix was being digested by elastase in the absence of SLPI’s antiprotease activity, preventing closure of the wound. It now appears, however, that inhibition of proteases is only a small component of SLPI’s anti-inflammatory function, as numerous studies have demonstrated that SLPI can downregulate the expression of pro-inflammatory cytokines. The first evidence of this was obtained in a study showing that SLPI can inhibit the expression of tumor necrosis factor α (TNFα) in mouse macrophage cell lines treated with lipopolysaccharide (LPS; Jin et al. 1997). Exposure to LPS in vivo also induced SLPI expression in mouse primary macrophages and polymorphonuclear leukocytes, as well as the lung and spleen, which suggested that SLPI is widely upregulated in response to a pro-inflammatory stimulus. Similar results have been obtained in studies of lung and liver injury. In mice that underwent hepatic ischemia/reperfusion injury, the lung and liver displayed significant cellular injury and infiltration of neutrophils as indicated by increased levels of serum alanine aminotransferase and tissue myeloperoxidase, respectively (Lentsch et al. 1999a). Significant increases in serum TNFα and macrophage inflammatory protein-2 (MIP-2) were also observed in these animals, along with induction of SLPI mRNA and protein expression in the liver. When SLPI was administered intravenously prior to the induction of ischemia and at the start of reperfusion, levels of TNFα, MIP-2, alanine aminotransferase, and myeloperoxidase were all significantly reduced in both lung and liver. SLPI can also regulate the conversion of transforming growth factor β (TGFβ) to its active form. Elevated levels of active TGFβ were observed in the epidermis of Slpi –/– mice 2 days after cutaneous injury, and the application of either SLPI or anti-TGFβ to the injury site decreased TGFβ levels, reduced inflammation, and reversed the impaired wound healing response normally seen in these animals (Ashcroft et al. 2000). These observations suggested that TGFβ acted as a chemoattractive factor for infiltrating leukocytes, leading to increased inflammation within the lesion site and that SLPI inhibits this response.
SLPI and Cancer
The role of SLPI in cancer has been widely debated. Some studies have reported that SLPI can inhibit proliferation and induce apoptosis in tumor cells, but the majority of studies in this field have identified SLPI as a factor that promotes invasion and metastasis. One piece of information that is not in question is that SLPI is highly expressed by a variety of tumor cell lines, including T98G human glioblastoma cells, Lewis lung carcinoma 3LL-S-sc cells, SKOV3 ovarian cancer cells, and GILM2 breast carcinoma cells. High levels of SLPI have also been reported in primary tumors such as colon adenoma, gastric carcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, and non-small cell lung carcinoma. This elevated expression has been associated with increased aggressiveness of the disease. High levels of SLPI expression led to enhanced tumorigenicity and proliferation of Lewis lung carcinoma 3LL-S-sc cells in vitro and increased the formation of lung metastases when these cells were injected in vivo (Devoogdt et al. 2003). In a study of breast cancer invasion and metastasis, HC11 mammary epithelial cells expressing the cfms proto-oncogene displayed significant increases in SLPI mRNA expression and corresponding increases in invasiveness and metastatic potential (Kluger et al. 2004). Finally, the survival, proliferation, and invasiveness of Hey-A8 ovarian cancer cells were enhanced when SLPI was overexpressed in these cells (Devoogdt et al. 2009). These responses were unchanged when the Hey-A8 cells were stably transfected to express SLPI that lacked protease inhibitor activity, which suggests that this function is not required for the ability of SLPI to enhance invasion and metastasis. Because of the strong correlation between elevated SLPI and malignancy and due to its presence in readily accessible fluids such as saliva, blood, and mucus, SLPI is being actively investigated as a potential diagnostic and prognostic biomarker for cancer.
While no definitive mechanism has been identified, new information continues to emerge regarding the signaling events and morphological changes that occur when tumor cells are exposed to SLPI. It has been reported that SLPI can induce sinusoidal angiogenesis and vascular remodeling in mice inoculated with MCH66C8 mammary tumor cells and that this promotes metastasis to the lung (Sugino et al. 2007). A 2015 study published in Nature has now provided further support for a vascular link between SLPI and metastasis (Wagenblast et al. 2015). In a mouse model of mammary carcinoma, SLPI expression was strongly correlated with metastatic progression. It was found that SLPI induced differentiation of the tumor cells into endothelial-like cells, a phenomenon known as vascular mimicry, which resulted in the formation of extravascular networks that carried blood to the tumor’s hypoxic core. SLPI also acted as an anticoagulant within these networks, which enhanced perfusion of the tumor and facilitated intravasation and metastasis of the tumor cells (Wagenblast et al. 2015). SLPI has also been shown to influence the survival and proliferation of cancer cells through regulation of the growth factor progranulin. In ovarian cancer cells, SLPI bound directly to progranulin, protecting it from degradation by elastase, and this significantly increased the survival and growth of the cells (Simpkins et al. 2008). This effect was confirmed using siRNA knockdown of SLPI, which resulted in reduced progranulin levels and increased apoptosis in the SKOV3 ovarian cancer cell line. Similar results were obtained in a recent study of castration-resistant prostate cancer (Zheng et al. 2016). Once again, SLPI expression was highly elevated in both prostate cancer cell lines and patient samples, and there was a strong correlation between SLPI-progranulin binding and the proliferation of human LNCaP prostate cancer cells. SLPI also significantly reduced TNFα-induced apoptosis in these cells, and based on the results of experiments with SLPI siRNA, the authors proposed that SLPI suppressed the expression of pro-apoptotic genes associated with the p53 pathway. These included cyclin-dependent kinase inhibitor 1A, TP53, Bcl-2-binding component 3, GADD45G, and sestrin-2. While it was not investigated in the study, it is possible that SLPI may regulate the expression of these genes by binding to their promoters and blocking transcription in a manner similar to that described for TNFα, and this would represent a new and intriguing avenue of investigation for cancer researchers.
SLPI and the Central Nervous System
Until recently, SLPI had been studied almost exclusively in the context of inflammation, infectious disease, and cancer, but researchers are now examining SLPI’s effects in the central nervous system (CNS). One of the first studies in this area reported that elevated levels of SLPI were present in human patients following ischemic stroke (Iłżecka and Stelmasiak 2002). When compared to control subjects, SLPI was significantly increased in serum samples obtained from patients at 5 and 12 days after stroke, and this elevated expression was associated with poor neurological outcomes and increased lesion size. Wang et al. (2003) then determined that SLPI mRNA expression was significantly elevated in ipsilateral cerebral cortex 12 h after a unilateral middle cerebral artery occlusion (MCAO) in rats and that this elevated expression was maintained for 5 days after the insult. Immunohistochemical staining for SLPI was present in neurons and astrocytes (but not macrophages or microglia) at 24 and 72 h after MCAO, which indicated that SLPI protein was expressed in response to ischemia. No induction of SLPI expression was observed in non-ischemic cortex or sham controls. Most importantly, rats that received intracortical injections of SLPI-expressing adenovirus 2 days prior to MCAO displayed improved neurological function and significant reductions in cortical lesion volume as measured by tetrazolium chloride staining. This suggested that SLPI is neuroprotective, and it has been proposed that SLPI is upregulated in an attempt to limit neuroinflammation and ischemic damage.
SLPI has been shown to have similar effects and patterns of expression in models of CNS injury. SLPI mRNA levels were dramatically increased 1 day after a moderate thoracic spinal cord contusion in mice and remained elevated until 7 days after injury (Ghasemlou et al. 2010). The amount of SLPI within the lesion site was also visibly higher when assessed by Western blot, reaching a maximum of 3 days after injury, and immunohistochemistry revealed that the protein was present in neurons, astrocytes, and neutrophils. To determine if SLPI could enhance functional recovery, wild-type mice underwent contusion injury and the animals then received daily intraperitoneal injections of SLPI for 7 days. When compared to mice that received saline, SLPI-treated mice showed significant improvement in locomotion over a 28-day period as measured by the Basso Mouse Scale. The spinal cords of these animals also displayed morphological changes that were indicative of neuroprotection, including increased tissue sparing and myelination, significant increases in the number of ventral horn motor neurons, and greater density of serotonergic axons caudal to the lesion site. In a subsequent study examining axonal regeneration, it was shown that SLPI can overcome the inhibitory effects of the CNS myelin protein’s myelin-associated glycoprotein (MAG) and Nogo and enhance neurite outgrowth for several different neuronal populations in vitro (Hannila et al. 2013). Following optic nerve crush in rats, regeneration of retinal ganglion cell axons was significantly increased when the animals received intravitreal injections of SLPI, and this was the first evidence that in vivo delivery of exogenous SLPI can promote regeneration of transected CNS axons. Mechanistically, administration of SLPI following spinal cord contusion resulted in increased expression of IκBα and decreased TNFα levels within the tissue, which provides further evidence that SLPI inhibits the expression of pro-inflammatory cytokines by modulating NF-κB activity (Ghasemlou et al. 2010). In the regeneration study, it was shown that SLPI localizes to the nuclei of neurons, binds to the promoter for Smad2 – a TGFβ signaling protein – and decreases Smad2 protein levels, leading to enhanced axonal growth (Hannila et al. 2013). Based on these findings and those of the cerebral ischemia studies, it appears that SLPI promotes neuronal survival and regeneration by downregulating the expression of genes that negatively affect these processes.
In the last 7 years, several groups have started to explore the role of SLPI in neurodegenerative diseases such as multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD). Given its ability to suppress inflammation, it is not surprising that SLPI has been studied in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. SLPI was highly expressed in neurons, astrocytes, and microglia in all three phases of EAE, and intriguingly, SLPI also stimulated the proliferation and differentiation of neural stem cells into oligodendrocytes, which suggests that it could be capable of facilitating remyelination (Mueller et al. 2008). In the G93A mouse model of ALS, high levels of SLPI were present in the CNS and skeletal muscle of the animals, and the progression of the disease coincided with increased expression of SLPI in skeletal muscle (Lilo et al. 2013). Elevated levels of SLPI were also present in bone marrow mesenchymal stem cells obtained from patients with sporadic ALS, leading the authors to propose that SLPI could potentially serve as an ALS biomarker (Lilo et al. 2013). The newest development in this field has come from a study of an Italian FTLD cohort in which the affected patients were carriers of progranulin mutations (Ghidoni et al. 2014). As noted above, SLPI interacts directly with progranulin, and among these patients, the individuals with the highest plasma levels of SLPI showed a delay in the onset of the disease. This led to the conclusion that SLPI regulates the penetrance of this phenotype, and it raises the possibility that there may be a correlation between SLPI expression and the development of other inherited forms of dementia.
SLPI has always been regarded as a protease inhibitor, but it is now apparent that its biological function is considerably more complex. As research into this protein has expanded from basic biochemistry to studies of systemic disease, one observation that has been consistently reported is that SLPI regulates the expression of genes. The binding of SLPI to DNA underlies its ability to reduce inflammation and promote axonal regeneration and could also account for its antibacterial effects and enhancement of metastasis. It is therefore likely that SLPI regulates the expression of many other genes within the mammalian genome and affects a variety of physiological and pathophysiological processes. As we learn more about these effects, our perception of this protein will undoubtedly continue to change, and in the future, perhaps SLPI will be known primarily as a transcriptional regulator.
- Antoniades CG, Khamri W, Abeles RD, Taams LS, Triantafyllou E, Possamai LA, Bernsmeier C, Mitry RR, O’Brien A, Gilroy D, Goldin R, Heneghan M, Heaton N, Jassem W, Bernal W, Vergani D, Ma Y, Quaglia A, Wendon J, Thursz M. Secretory leukocyte protease inhibitor: a pivotal mediator of anti-inflammatory responses in acetaminophen-induced acute liver failure. Hepatology. 2014;59:1564–76.PubMedCrossRefGoogle Scholar
- Foth BJ, Tsai IJ, Reid AJ, Bancroft AJ, Nichol S, Tracey A, Holroyd N, Cotton JA, Stanley EJ, Zarowiecki M, Liu JZ, Huckvale T, Cooper PJ, Grencis RK, Berriman M. Whipworm genome and dual-species transcriptome analyses provide molecular insights into an intimate host-parasite interaction. Nat Genet. 2014;46:693–700.PubMedPubMedCentralCrossRefGoogle Scholar
- Gomez SA, Argüelles CL, Guerrieri D, Tateosian NL, Amiano NO, Slimovich R, Maffia PC, Abbate E, Musella RM, Garcia VE, Chuluyan HE. Secretory leukocyte protease inhibitor: a secreted pattern recognition receptor for mycobacteria. Am J Respir Crit Care Med. 2009;179:247–53.PubMedCrossRefGoogle Scholar
- Hannila SS, Siddiq MM, Carmel JB, Hou J, Chaudhry N, Bradley PMJ, Hilaire M, Richman EL, Hart RP, Filbin MT. Secretory leukocyte protease inhibitor reverses inhibition by CNS myelin, promotes regeneration in the optic nerve, and suppresses expression of the TGFβ signaling protein Smad2. J Neurosci. 2013;33:5138–51.PubMedPubMedCentralCrossRefGoogle Scholar
- Py B, Basmaciogullari S, Bouchet J, Zarka M, Moura IC, Benhamou M, Monteiro RC, Hocini H, Madrid R, Benichou S. The phospholipid scramblases 1 and 4 are cellular receptors for the secretory leukocyte protease inhibitor and interact with CD4 at the plasma membrane. PLoS One. 2009;4:e5006.PubMedPubMedCentralCrossRefGoogle Scholar
- Schneeberger S, Hautz T, Wahl SM, Brandacher G, Sucher R, Steinmassl O, Steinmassl P, Wright CD, Obrist P, Werner ER, Mark W, Troppmair J, Margreiter R, Amberger A. The effect of secretory leukocyte protease inhibitor (SLPI) on ischemia/reperfusion injury in cardiac transplantation. Am J Transplant. 2008;8:773–82.PubMedCrossRefGoogle Scholar
- Wagenblast E, Soto M, Gutiérrez-Ángel S, Hartl CA, Gable AL, Maceli AR, Erard N, Williams AM, Kim SY, Dickopf S, Harrell JC, Smith AD, Perou CM, Wilkinson JE, Hannon GJ, Knott SR. A model of breast cancer heterogeneity reveals vascular mimicry as a driver of metastasis. Nature. 2015;520:358–62.PubMedPubMedCentralCrossRefGoogle Scholar
- Wang X, Li X, Xu L, Zhan Y, Yaish-Ohad S, Erhardt JA, Barone FC, Feuerstein GZ. Up-regulation of secretory leukocyte protease inhibitor (SLPI) in the brain after ischemic stroke: adenoviral expression of SLPI protects brain from ischemic injury. Mol Pharmacol. 2003;64:833–40.PubMedCrossRefGoogle Scholar