Cementoin-SLPI fusion protein binds to human monocytes and epithelial cells and shows higher biological activity than SLPI.

Secretory Leukocyte Proteinase Inhibitor (SLPI) is an antiinflammatory peptide that blocks the activity of serine proteases, primarily the neutrophil elastase. In an attempt to direct the activity of SLPI on inflamed sites, a chimera consisting of the transglutaminase II substrate domain of trappin 2 (cementoin), and the mature SLPI protein was constructed. Cell attachment and biological activity were compared between SLPI and this chimera. By using whole cell ELISA, fluorescence microscopy and flow cytometry assays we observed that the cementoin-SLPI fusion protein (FP) but not SLPI attached to a human lung epithelial cell line and monocytes. A maximum attachment was achieved 15 min after FP was added to the cell cultures. In an elastase activity assay, we observed that FP retained its antiprotease activity and that at equimolar amount of proteins, FP was more efficient than SLPI in the inhibition. Both, FP and SLPI inhibits IL-2-induced lymphocyte proliferation, however, lower amounts of FP were required to achieve this inhibition. Furthermore, FP binds to mycobacteria and maintained the bactericidal activity observed for SLPI. Overall, these results show that this new chimera is able to attach to the cell surfaces retaining and improving some biological activities described for SLPI.

Anti-tumor effect of SLPI on mammary but not colon tumor growth.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that was related to cancer development and metastasis dissemination on several types of tumors. However, it is not known the effect of SLPI on mammary and colon tumors. The aim of this study was to examine the effect of SLPI on mammary and colon tumor growth. The effect of SLPI was tested on in vitro cell apoptosis and in vivo tumor growth experiments. SLPI over-expressing human and murine mammary and colon tumor cells were generated by gene transfection. The administration of murine mammary tumor cells over-expressing high levels of SLPI did not develop tumors in mice. On the contrary, the administration of murine colon tumor cells over-expressing SLPI, developed faster tumors than control cells. Intratumoral, but not intraperitoneal administration of SLPI, delayed the growth of tumors and increased the survival of mammary but not colon tumor bearing mice. In vitro culture of mammary tumor cell lines treated with SLPI, and SLPI producer clones were more prone to apoptosis than control cells, mainly under serum deprivation culture conditions. Herein we demonstrated that SLPI induces the apoptosis of mammary tumor cells in vitro and decreases the mammary but not colon tumor growth in vivo. Therefore, SLPI may be a new potential therapeutic tool for certain tumors, such as mammary tumors.

Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4(+) lymphocyte proliferation.

Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti-inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [(3) H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin-2 (IL-2) or OKT3 monoclonal antibodies in a dose-dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI-treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL-4, IL-6 and IL-10 in the cell culture supernatants. SLPI-treated monocyte culture supernatant inhibited the CD4(+) lymphocyte proliferation but did not affect the proliferation of CD8(+) cells. Moreover, IL-2 increased T-bet expression and the presence of SLPI significantly decreased it. Finally, SLPI-treated monocyte culture supernatant dramatically decreased interferon-γ but increased IL-4, IL-6 and IL-10 in the presence of IL-2-treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response.

Cellular origin of the Bufo arenarum sperm receptor gp75, a ZP2 family member: its proteolysis after fertilization.

BACKGROUND INFORMATION: The egg envelope is an extracellular matrix that surrounds oocytes. In frogs and mammals, a prominent feature of envelope modification following fertilization is the N-terminal proteolysis of the envelope glycoproteins, ZPA [ZP (zona pellucida) A]. It was proposed that ZPA N-terminal proteolysis leads to a conformational change in egg envelope glycoproteins, resulting in the prevention of polyspermy. Bufo arenarum VE (vitelline envelope) is made up of at least four glycoproteins: gp120 (glycoprotein 120), gp75, gp41 and gp38. The aim of the present study was to identify and characterize the baZPA (B. arenarum ZPA homologue). Also, our aim was to evaluate its integrity and functional significance during fertilization. RESULTS: VE components were labelled with FITC in order to study their sperm-binding capacity. The assay showed that gp75, gp41 and gp38 possess sperm-binding activity. We obtained a full-length cDNA of 2062 bp containing one ORF (open reading frame) with a sequence for 687 amino acids. The predicted amino acid sequence had close similarity to that of mammalian ZPA. This result indicates that gp75 is the baZPA. Antibodies raised against an N-terminal sequence recognized baZPA and inhibited sperm-baZPA extracted from VE binding. This protein does not induce the acrosome reaction in homologue sperm. Northern-blot studies indicated that the transcript is exclusively expressed in the ovary. In situ hybridization studies confirmed this and pointed to previtellogenic oocytes and follicle cells surrounding the oocyte as the source of the transcript. baZPA was cleaved during fertilization and the N-terminal peptide fragment remained disulfide bonded to the glycoprotein moiety following proteolysis. CONCLUSION: From the sequence analysis, it was possible to consider that gp75 is the baZPA. It is expressed by previtellogenic oocytes and follicle cells. Also, it can be considered as a sperm receptor that undergoes N-terminal proteolysis during fertilization. The N-terminal peptide could be necessary for sperm binding.