Bellinda A. Bladergroen

The Granzyme B Inhibitor, Protease Inhibitor 9, Is Mainly Expressed by Dendritic Cells and at Immune-Privileged Sites

Granzyme B is released from CTLs and NK cells and an important mediator of CTL/NK-induced apoptosis in target cells. The human intracellular serpin proteinase inhibitor (PI)9 is the only human protein able to inhibit the activity of granzyme B. As a first step to elucidate the physiological role of PI9, PI9 protein expression in various human tissues was studied. A mAb directed against human PI9 was developed, which specifically stained PI9-transfected COS-7 cells, and was used for immunohistochemistry. Both in primary lymphoid organs and in inflammatory infiltrates, PI9 was present in different subsets of dendritic cells. Also T-lymphocytes in primary and organ-associated lymphoid tissues were PI9 positive. Endothelial cells of small vessels in most organs tested as well as the endothelial layer of large veins and arteries showed strong PI9 staining. Surprisingly, high PI9 protein expression was also found at immune-privileged sites like the placenta, the testis, the ovary, and the eye. These data fit with the hypothesis that PI9 is expressed at sites where degranulation of CTL or NK cells is potentially deleterious.

Expression of the apoptosis inhibitor protease inhibitor 9 predicts clinical outcome in vaccinated patients with stage III and IV melanoma

Purpose: There have been reports of successful treatment of metastatic melanoma patients with active specific immunotherapy (ASI) using irradiated autologous tumor cell vaccination. It is still unknown why some patients respond and others do not. Tumor cells can evade the immune system, for example through interference with antigen presentation by down-regulation of MHC molecules or expressing proteins interfering with cytotoxic lymphocyte–induced apoptosis like the granzyme B antagonist protease inhibitor 9 (PI-9). Experimental Design: PI-9 expression was detected in melanoma cell lines. To investigated if PI-9 is important in the response to ASI, paraffin-embedded tissues from stage III or IV melanoma patients were stained. Results: PI-9 is expressed in melanoma cells and expression in metastasized melanoma cells is, in this group of patients, an adverse prognostic marker with regard to overall and disease-free survival. Moreover, loss of MHC-1 expression frequently occurs during tumor progression but is not associated with poor clinical outcome. Interestingly, melanoma patients with a favorable clinical outcome after ASI therapy usually have high percentages of activated (granzyme B–positive) tumor-infiltrating lymphocytes at time of first diagnosis and low percentages of activated lymphocytes at time of recurrent tumor. Conclusions: Expression of PI-9 in metastatic melanoma cells is associated with unfavorable clinical outcome whereas MHC-1 down-regulation is not. Although it cannot be proven that PI-9 expression is directly responsible for failure of immunotherapy, these data suggest that expression of PI-9 could be an important immune escape mechanism and that modulation of this inhibitor may enhance the efficacy of immunotherapy.

In Vivo Recruitment of Hematopoietic Cells Using Stromal Cell–Derived Factor 1 Alpha–Loaded Heparinized Three-Dimensional Collagen Scaffolds

Implantable three-dimensional (3D) constructs to engineer tissue have great therapeutic potential in regenerative medicine and immunotherapy. However, autonomous recruitment of cells into the engineered scaffold in vivo is hampered by lack of attracting scaffolds. As a first step to engineering immune tissue, 3D collagen scaffolds were investigated for their ability to enhance in vivo recruitment and growth of various hematopoietic cells. Scaffolds containing immobilized heparin to trap the stem cell chemo-attractant stromal cell-derived factor 1 alpha (SDF1alpha) were implanted subcutaneously into C57Bl6 mice, and influx of cells was monitored using immunohistochemistry. Five weeks post-implantation, heparinized scaffolds were always populated by cells, but incorporating SDF1alpha considerably stimulated recruitment of cells. SDF1alpha could not exert this effect when the formation of a SDF1alpha gradient was abrogated. Scaffolds were mainly populated by CD11b+ and CD11c+ myeloid cells and fibroblasts. One week after implantation, scaffolds harbored only low numbers of cells. Apparently, not all CXCR4-expressing cells, like large numbers of granulocytes, migrate into the scaffold, but retransplantation of a 1-week-old scaffold from a CD45.2(+) into a CD45.1(+) mouse yielded a scaffold harboring mainly CD45.2(+) cells after 5 weeks. These data confirm that only a few progenitor cells are recruited early after implantation. These cells then proliferate and differentiate along different lineages and determine the outcome after 5 weeks.

Distribution of the human intracellular serpin protease inhibitor 8 in human tissues

Ovalbumin-like serine protease inhibitors are mainly localized intracellularly and their in vivo functions are largely unknown. To elucidate their physiological role(s), we studied the expression of one of these inhibitors, protease inhibitor 8 (PI-8), in normal human tissues by immunohistochemistry using a PI-8-specific monoclonal antibody. PI-8 was strongly expressed in the nuclei of squamous epithelium of mouth, pharynx, esophagus, and epidermis, and by the epithelial layer of skin appendages, particularly by more differentiated epithelial cells. PI-8 was also expressed by monocytes and by neuroendocrine cells in the pituitary gland, pancreas, and digestive tract. Monocytes showed nuclear and cytoplasmic localization of PI-8, whereas neuroendocrine cells showed only cytoplasmic staining. In vitro nuclear localization of PI-8 was confirmed by confocal analysis using serpin-transfected HeLa cells. Furthermore, mutation of the P1 residue did not affect the subcellular distribution pattern of PI-8, indicating that its nuclear localization is independent of the interaction with its target protease. We conclude that PI-8 has a unique distribution pattern in human tissues compared to the distribution patterns of other intracellular serpins. Additional studies must be performed to elucidate its physiological role.

The granzyme B inhibitor proteinase inhibitor 9 (PI9) is expressed by human mast cells.

The activity of granzyme B, a main effector molecule of cytotoxic T lymphocytes (CTL) and natural killer cells, is regulated by the human intracellular serpin proteinase inhibitor 9 (PI9). This inhibitor is particularly expressed by CTL and dendritic cells, in which it serves to protect these cells against endogenous and locally released granzyme B. Moreover, PI9 expression by neoplastic cells may constitute one of the mechanisms for tumors to escape immune surveillance. Here we show that PI9 is also expressed by human mast cells. In immunohistochemical studies using a PI9‐specific monoclonal antibody, strong cytoplasmic staining for PI9 was found in normal mast cells in various tissues throughout the body. In addition, in 80% of all cases of cutaneous and systemic mastocytosis tested the majority of the mast cells expressed PI9. As an in vitro model for PI9 expression by mast cells, we studied expression by the human mast cell line HMC‐1. Stimulation of HMC‐1 with PMA and the calcium ionophore A23187 resulted in a marked increase of PI9 expression. Thus, PI9 is expressed by activated mast cells. We suggest that this expression serves to protect these cells against apoptosis induced by granzyme B released during initiation of the local inflammatory response.

Production, characterization, and use of serpin antibodies.

Serine protease inhibitors (serpins) constitute a still expanding superfamily of structural similar proteins, which are localized extracellularly and intracellularly. Serpins play a central role in the regulation of a wide variety of (patho) physiological processes including coagulation, fibrinolysis, inflammation, development, tumor invasion, and apoptosis. Serpins have a unique mechanism of inhibition that involves a profound change in conformational state upon interaction with their protease. This conformational change enables the production of monoclonal antibodies specific for native, complexed, and inactivated serpins. Antibodies, and assays based on these antibodies, have been helpful in elucidating the (patho) physiological function of serpins in the last decade. Serpin-specific antibodies can be used for: (1) structure-function studies such as detection of conformational changes; (2) identification of target-proteases; (3) the detection and quantification of serpin and serpin-protease complexes in bodily fluids by immunoassays such as ELISA, RIA or FACS; (4) detection of serpins in tissues by immunohistochemistry; and (5) possible therapeutical interventions. This review summarizes the techniques we have used to obtain and screen antibodies against extra- and intracellular serpins, as well as the use of these antibodies for some of the above-mentioned purposes.