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.

Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells.

IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), CD80, CD206, and CD1a. Monocytes stained positive with Abs against heparan sulfate (HS) and chondroitin sulfate (CS) B (CSB; dermatan sulfate), but not with Abs that recognize CSA, CSC, and CSE. Inhibition of sulfation of monocyte/DC cell surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing single-chain Abs. This correlated with hampered IL-4-induced DC differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell surface GAGs support IL-4 activity. Furthermore, removal of cell surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD, and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O sulfates (CSB and heparin). Together, these data demonstrate that monocyte cell surface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs.

Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation.

Mast cells are widely distributed throughout the body and express effector functions in allergic reactions, inflammatory diseases, and host defense. Activation of mast cells results in exocytosis of preformed chemical mediators and leads to novel synthesis and secretion of lipid mediators and cytokines. Here, we show that human mast cells also express and release the cytotoxic lymphocyte-associated protease, granzyme B. Granzyme B was active and localized in cytoplasmic granules, morphologically resembling those present in cytotoxic lymphocytes. Expression and release of granzyme B by mast cell-lines HMC-1 and LAD 2 and by cord blood- and mature skin-derived human mast cells depended on the mode of activation of these cells. In mast cell lines and cord blood-derived mast cells, granzyme B expression was mainly induced by non-physiological stimuli (A23187/PMA, Compound 48/80) and substance P. In contrast, mature skin-derived mast cells only produced granzyme B upon IgE-dependent stimulation. We conclude that granzyme B is expressed and released by human mast cells upon physiologic stimulation. This suggests a role for granzyme B as a novel mediator in mast cell biology.

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.

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.

Intracellular serpin SERPINB6 (PI6) is abundantly expressed by human mast cells and forms complexes with beta-tryptase monomers.

SERPINB6 (PI6) is a member of the intracellular serine protease inhibitors (serpins). Previous studies showed that SERPINB6 is localized mainly in the cytoplasm of endothelial cells, some epithelial cells, monocytes, and neutrophils. In these cells SERPINB6 is thought to prevent cellular damage by scavenging leaking lysosomal proteases. We show here, using novel, well-defined monoclonal antibodies, that SERPINB6 is abundantly expressed by mast cells in all organs and by the human mast cell line HMC-1. Gel filtration experiments revealed that the latter cells contain a high-molecular-weight form of SERPINB6, which consists of sodium dodecyl sulfate (SDS)-stable complexes of this inhibitor with monomeric beta-tryptase. Expression of SERPINB6 by mast cells was compared with those of tryptase and CD117 (c-kit) in biopsies from patients with different forms of mast cell disease. In all cases the lesional mast cells expressed SERPINB6, and, in diffuse cutaneous mastocytosis and mastocytoma, SERPINB6 was expressed by a substantially higher number of mast cells when compared with tryptase. In conclusion, SERPINB6 is abundantly expressed by normal mast cells and by mast cells in mastocytoma lesions. We suggest that in mast cells, SERPINB6 serves to regulate the activity of endogenous beta-tryptase in the cytoplasm.

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 P(1) 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.

Expression levels of apoptosis-related proteins predict clinical outcome in anaplastic large cell lymphoma.

In vitro studies suggest that resistance to chemotherapy-induced apoptosis might explain poor response to therapy in fatal cases. Actual execution of apoptosis depends on proper functioning of effector caspases, particularly caspase 3, and on the expression levels of apoptosis-regulating proteins, including Bcl-2 and the recently identified granzyme B- specific protease inhibitor 9 (PI9). Thus, high levels of caspase 3 activation should reflect proper functioning of the apoptosis pathways, resulting in chemotherapy-sensitive neoplastic cells and a favorable prognosis. We tested this hypothesis by quantifying numbers of tumor cells positive for active caspase 3, Bcl-2, and PI9, respectively, in pretreatment biopsies of systemic anaplastic large cell lymphoma (ALCL) patients and by comparing these numbers with clinical outcome. Activation of caspase 3 in more than 5% of the tumor cells was strongly correlated with a highly favorable outcome. High numbers of Bcl-2- and PI9-positive tumor cells were found to predict unfavorable prognosis. This prognostic effect was strongly related to anaplastic lymphoma kinase (ALK) status: ALK-positive ALCL had significantly higher levels of active caspase 3, while high expression of the antiapoptotic proteins Bcl-2 and PI9 was almost completely restricted to ALK-negative cases. In conclusion, high numbers of active caspase 3-positive tumor cells predict a highly favorable prognosis in systemic ALCL patients. Poor prognosis is strongly related to high numbers of Bcl-2- and PI9-positive neoplastic cells. These data support the notion that a favorable response to chemotherapy depends on an intact apoptosis cascade. Moreover, these data indicate that differences in prognosis between ALK-positive and ALK-negative ALCL might be explained by differences in expression of apoptosis-inhibiting proteins.

Expression of the granzyme B inhibitor, protease inhibitor 9, by tumor cells in patients with non-Hodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the immune system?

In tumor cells, the serine protease granzyme B is the primary mediator of apoptosis induced by cytotoxic T lymphocytes (CTLs)/natural killer (NK) cells. The human intracellular serpin proteinase inhibitor 9 (PI9) is the only known human protein able to inhibit the proteolytic activity of granzyme B. When present in the cytoplasm of T lymphocytes, PI9 is thought to protect CTLs against apoptosis induced by their own misdirected granzyme B. Based on the speculation that tumors may also express PI9 to escape CTL/NK cell surveillance, immunohistochemical studies on the expression of PI9 in various lymphomas were performed. Ninety-two cases of T-cell non-Hodgkin lymphoma (NHL), 75 cases of B-cell NHL, and 57 cases of Hodgkin lymphomas were stained with a PI9-specific monoclonal antibody. In T-cell NHL, highest PI9 expression was found in the extranodal T-cell NHL. In nearly 90% of enteropathy-type T-cell NHLs and 80% of NK/T-cell, nasal-type lymphomas, the majority of the tumor cells expressed PI9. In nodal T-anaplastic large cell lymphomas and peripheral T-cell lymphomas (not otherwise specified), PI9 expression occurred less frequently. In B-cell NHL, PI9 expression was associated with high-grade malignancy; 43% of diffuse large B-cell lymphomas showed PI9(+) tumor cells. Finally, PI9 expression was also found in 10% of Hodgkin lymphomas. This is the first report describing the expression of the granzyme B inhibitor PI9 in human neoplastic cells in vivo. Expression of this inhibitor is yet another mechanism used by tumor cells to escape their elimination by cytotoxic lymphocytes.