Localization of heparanase in normal and pathological human placenta

Degradation of extracellular matrix (ECM) components is critical for invasion. Heparan sulphate proteoglycans are abundant in the ECM of the placenta and the decidua, hence their degradation may disassemble the matrix and facilitate placentation and trophoblast invasion. This study investigates the expression of heparanase in normal and pathological placentation using RT-PCR, in-situ hybridization and immunohistochemistry analysis to detect heparanase in specific cells of the placenta and at the fetal-maternal interface throughout pregnancy. Heparanase was observed in villous cytotrophoblasts (CT), syncytial trophoblasts (ST) and in intermediate trophoblast cell columns in normal first trimester, molar and ectopic pregnancies. The heparanase protein was preferentially expressed in the endothelium of fetal capillaries, and to a much lesser extent in larger fetal vessels. Extravillous trophoblasts (EVT) invading the decidua and the maternal vessels were also heparanase positive. In the second and third trimesters, villous CT remained heparanase positive whereas ST showed variable heparanase expression. EVT invading the placental implantation site were also positively stained. A similar pattern was observed in samples obtained from pre-eclamptic placentae and from placenta accreta. Our results indicate consistent expression of heparanase in normal and abnormal placenta, in small fetal vessels and in a variety of trophoblast subpopulations with different invasive potentials.     

Gestational Age-Dependent Extravillous Cytotrophoblast Osteopontin Immunolocalization Differentiates Between Normal and Preeclamptic Pregnancies

PROBLEM: Normal placentation requires modulation of proliferative cytotrophoblast to an invasive phenotype. Preeclampsia is characterized by failed cytotrophoblast invasion and arterial remodeling. Osteopontin (OPN) is an extracellular matrix protein implicated in cell adhesion, spreading, and invasion. METHOD OF STUDY: To investigate gestational age-related OPN expression, placental immunostaining was performed. To investigate the role of OPN in uteroplacental vascular pathology, placental immunostaining from pregnancies with preeclampsia (n = 12), fetal growth retardation (FGR) (n = 8), or both (n = 4) was compared with gestational age-matched controls (n = 24). RESULTS: In non-preeclamptic pregnancies, OPN immunolocalized to basal plate and intervillous cytotrophoblasts from 24–30 weeks (n = 13). In preeclampsia, OPN immunoreactivity was detected from 24–40 weeks. Cytotrophoblasts from FGR placentas were OPN-positive until 30 weeks, unless preeclampsia accompanied the FGR. In this case, cytotrophoblasts were OPN-positive from 24–40 weeks. CONCLUSIONS: The data suggest a role for OPN in cytotrophoblast invasion of the maternal vasculature/extracellular matrix during non-preeclamptic placentation, and OPN may serve as a marker for placental bed remodeling.     

The impact of heparanese and heparin on cancer metastasis and angiogenesis

Heparan sulfate (HS) proteoglycans play a key role in the self-assembly, insolubility and barrier properties of the extracellular matrix (ECM). Cleavage of HS therefore affects the integrity of tissues and hence normal and pathological phenomena involving cell migration and response to changes in the ECM. Mammalian heparanase, HS-degrading endoglycosidase,is synthesized as a latent 65 kDa precursor that undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa subunits that heterodimerize to form a highly active enzyme. Heparanase is preferentially expressed in human tumors and its over-expression in tumor cells confers an invasive phenotype in experimental animals. Heparanase also releases angiogenic factors from the ECM and tumor micro environment and thereby induces an angiogenic response in vivo. Enhanced heparanase expression correlates with metastatic potential, tumor vascularity and reduced postoperative survival of cancer patients.Heparanase also promotes cell adhesion, survival and signaling events, independent of its enzymatic activity. These observations, the anti-cancerous effect of heparanase gene silencing and of heparanase inhibiting molecules as well as the unexpected identification of a predominant functional heparanase, suggest that the enzyme is a promising target for anti-cancer drug development. Here, we summarize recent progress in molecular and cellular aspects of heparanase, emphasizing its causal involvement in cancer metastasis and angiogenesis, and discuss the development of heparin-like heparanase inhibitors.     

Human Trophoblast Cell Adhesion to Extracellular Matrix Protein,Entactin

PROBLEM: Trophoblast interaction with endometrial extracellular matrix (ECM) is crucial during human embryo implantation and placentation. Entactin, a ubiquitous basement membrane glycoprotein, plays a central role in ECM assembly, cell attachment, and chemotaxis. The present study was conducted to examine the possible role of entactin in promoting human trophoblast adhesion. METHODS: Using an extended life span first trimester trophoblast cell line HTR-8/SVneo (HTR) and a cell adhesion assay, we measured the adherence of human first trimester trophoblasts to recombinant entactin and its domains. Also, we used flow cytometry and indirect immunofluorescence to detect the presence of integrins that may be involved in human trophoblast-entactin interaction; these methods were used to analyze HTR cells, as well as tissue sections and freshly isolated human trophoblasts from first trimester and term placenta. RESULTS: We found that first trimester trophoblast cells were highly adherent to entactin and its E and G2 domains but not to G1 or G3 domains. Using indirect immunofluorescence and flow cytometry, we found that both β1 and β3 integrin subunits were expressed on the surface of HTR trophoblast cells adhering to entactin; in contrast, β2 and β4 integrin subunits were not detected. In addition, we found that αvβ3 was expressed on freshly isolated villous cytotrophoblasts and cytotrophoblast and syncytiotrophoblasts in tissue sections from term placenta. The β3 integrin subunit was expressed in cytotrophoblasts and syncytiotrophoblasts in villi of first trimester placental tissue sections. CONCLUSION: Recombinant entactin promotes human trophoblast cell adhesion through both its E and G2 domains and these specific adhesive interactions may be mediated by β1 and/or β3 class integrins.     

Plasma Membrane-Associated pY397FAK Is a Marker of Cytotrophoblast Invasion in Vivo and in Vitro

During human pregnancy specialized placental cells of fetal origin, termed cytotrophoblasts, invade the uterus and its blood vessels. This tumor-like process anchors the conceptus to the mother and diverts the flow of uterine blood to the placenta. Previously, we showed that the expression of molecules with important functional roles, including a number of extracellular matrix integrin receptors, is precisely modulated during cytotrophoblast invasion in situ. Here we exploited this observation to study the role of the focal adhesion kinase (FAK), which transduces signals from the extracellular matrix and recruits additional signaling proteins to focal adhesions. Immunolocalization studies on tissue sections showed that FAK is expressed by cytotrophoblasts in all stages of differentiation. Because extracellular matrix-induced integrin clustering results in FAK (auto)phosphorylation on tyrosine 397 (Y397FAK), we also localized this form of the molecule. Immunolocalization experiments detected Y397FAK in a subset of cytotrophoblasts near the surface of the uterine wall. To assess the functional relevance of this observation, we used an adenovirus strategy to inhibit cytotrophoblast expression of FAK as the cells differentiated along the invasive pathway in vitro. Compared to control cells transduced with a wild-type virus, cytotrophoblasts that expressed antisense FAK exhibited a striking reduction in their ability to invade an extracellular matrix substrate. When cytotrophoblast differentiation was compromised (hypoxia in vitro, preeclampsia in vivo), Y397FAK levels associated with the plasma membrane were strikingly lower, although total FAK levels did not change. Together our results suggest that (auto)phosphorylation of Y397 on FAK is a critical component of the signaling pathway that mediates cytotrophoblast migration/invasion.     

Mice deficient in heparanase exhibit impaired dendritic cell migration and reduced airway inflammation

Heparanase is a β‐d ‐endoglucuronidase that cleaves heparan sulphate, a key component of the ECM and basement membrane. The remodelling of the ECM by heparanase has been proposed to regulate both normal physiological and pathological processes, including wound healing, inflammation, tumour angiogenesis and cell migration. Heparanase is also known to exhibit non‐enzymatic functions by regulating cell adhesion, cell signalling and differentiation. In this study, constitutive heparanase‐deficient (Hpse^?/?) mice were generated on a C57BL/6 background using the Cre/loxP recombination system, with a complete lack of heparanase mRNA, protein and activity. Although heparanase has been implicated in embryogenesis and development, Hpse^?/? mice are anatomically normal and fertile. Interestingly, consistent with the suggested function of heparanase in cell migration, the trafficking of dendritic cells from the skin to the draining lymph nodes was markedly reduced in Hpse^?/? mice. Furthermore, the ability of Hpse^?/? mice to generate an allergic inflammatory response in the airways, a process that requires dendritic cell migration, was also impaired. These findings establish an important role for heparanase in immunity and identify the enzyme as a potential target for regulation of an immune response.     

Subcellular localization of heparanase in human neutrophils.

The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel filtration analysis of 35S-labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECM-derived, heparan sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.     

Heparanase upregulation by colonic epithelium in inflammatory bowel disease.

Heparanase is an endo-beta-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, yielding HS fragments of still appreciable size ( approximately 5-7 kDa). Heparanase activity has long been detected in a number of cell types and tissues. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the extracellular matrix barrier. Similarly, heparanase activity was implicated in neovascularization, inflammation and autoimmunity, involving migration of vascular endothelial cells and activated cells of the immune system. The involvement of heparanase in inflammatory processes of the gastrointestinal tract has not been examined. Here, we utilized immunohistochemical analysis to investigate heparanase expression in acute and chronic inflammatory conditions. Heparanase expression was not detected in specimens derived from normal colon tissue. In contrast, strong heparanase staining was observed in Crohn's disease and ulcerative colitis, but not in infectious colitis. Interestingly, heparanase staining was primarily observed in epithelial rather than immune cells. Importantly, un-fractionated as well as low molecular weight heparin (enoxaparin), which exhibit a strong inhibitory activity towards heparanase, have proven efficacious in ulcerative colitis and Crohn's disease patients, suggesting that heparanase is actively involved in these pathologies and thus may be considered as a target for the development of anti-inflammatory therapies.     

Preeclampsia Is Associated with Widespread Apoptosis of Placental Cytotrophoblasts within the Uterine Wall

Preeclampsia is a serious pregnancy complication diagnosed by signs of widespread maternal endothelial dysfunction. In normal pregnancy, a subpopulation of placental cytotrophoblast stem cells executes an unusual differentiation program that leads to invasion of the uterus and its vasculature. This process attaches the conceptus to the uterine wall and starts the flow of maternal blood to the placenta. Preeclampsia is associated with abnormal cytotrophoblast differentiation, shallow invasion, and decreased blood flow to the placenta. To determine whether abnormal differentiation and/or hypoxia leads to cytotrophoblast apoptosis, we used the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) method to label DNA strand breaks in tissue sections of the placenta and the uterine wall to which it attaches. Control samples (n = 9) showed almost no apoptosis, but in samples from patients with preeclampsia, 15-50% of the cytotrophoblasts that invaded the uterine wall were labeled (8/9 samples). These same cells failed to stain for Bcl-2, a survival factor normally expressed by trophoblasts in both the placenta and the uterine wall. Our results show that preeclampsia is associated with widespread apoptosis of cytotrophoblasts that invade the uterus. The magnitude of programmed cell death in this population may account for the sudden onset of symptoms in some patients, as well as the associated coagulopathies.     

Invasive cytotrophoblast apoptosis in pre-eclampsia

Pre-eclampsia is a serious pregnancy complication diagnosed by signs of widespread maternal endothelial dysfunction. In normal pregnancy, a subpopulation of placental cytotrophoblast stem cells executes a differentiation programme that leads to invasion of the uterus and its vasculature. This process attaches the conceptus to the uterine wall and starts the flow of maternal blood to the placenta. In pre-eclampsia, cytotrophoblasts fail to differentiate along the invasive pathway. The functional consequences of this abnormality negatively affect interstitial and endovascular invasion, thereby compromising blood flow to the maternal-fetal interface. To determine whether abnormal differentiation and/or hypoxia leads to apoptosis of invasive cytotrophoblasts, we used the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) method to label DNA strand breaks in tissue sections of the placenta and the uterine wall to which it attaches. Control samples (n = 9) showed little or no apoptosis in any location, but in samples from patients with pre-eclampsia, 15-50% of the cytotrophoblast subpopulation that invaded the uterine wall was labelled (8/9 samples). These same cells failed to stain for Bcl-2, a survival factor normally expressed by trophoblasts in both the placenta and the uterine wall. Our results show that pre-eclampsia is associated with widespread apoptosis of cytotrophoblasts that invade the uterus. The magnitude of programmed cell death in this population may account for the sudden onset of symptoms in some patients, as well as the associated coagulopathies.     

Heparanase expression during normal liver development and following partial hepatectomy

Heparan sulphate proteoglycans are major components of the liver extracellular matrix. Their cleavage by heparanase (endo-beta-glucuronidase) may thus be involved in liver-specific normal and pathological processes. Heparanase mRNA and protein were expressed during liver development but not in the mature healthy liver. A biphasic gain of heparanase expression, detected by immunostaining, western blotting, and real-time RT-PCR, was clearly noted following partial hepatectomy, peaking at 12 and 96-168 h and subsiding 2 weeks post-surgery. Expression of heparan sulphate gradually increased throughout the regeneration process. Unlike heparanase, baseline levels of matrix metalloproteinase-2 (MMP-2) were detected in the intact liver, increasing up to 4 days following partial hepatectomy and subsiding at day 10. Bands matching MMP-9 were absent prior to hepatectomy, but visible 2 h post-hepatectomy. Thioacetamide-induced liver fibrosis was associated with increased levels of MMP-9 and MMP-2, correlating with the severity of the disease. Elevated heparanase levels were noted in the early stages of fibrosis, with no further increase evident in rats exhibiting higher fibrotic grades. Taken together, these data suggest a role for heparanase during liver development and remodelling.     

Coexpression of heparanase, basic fibroblast growth factor and vascular endothelial growth factor in human esophageal carcinomas

Heparan sulfate (HS), which is degraded by heparanase, plays an important role in cell adhesion, insolubility of the extracellular matrix (ECM) and as a reservoir for various growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). In the present study, we examined the immunohistochemical expression of heparanase, bFGF and VEGF, and evaluated the correlation between their expression and microvessel density (MVD) in human esophageal carcinomas. Heparanase, bFGF and VEGF were immunolocalized predominantly to the carcinoma cells, but they were also localized to the endothelial cells of microvessels near the carcinoma cell nests. In carcinomas with invasion of the muscular layer or adventitia, heparanase staining was stronger at the invasive areas of carcinomas than the intraepithelial spread. Expression of heparanase and bFGF and the degree of MVD were associated with tumor invasion, lymph node metastasis and pathological stages. Cases with positive staining for heparanase, bFGF or VEGF tended to have a higher MVD than those without staining, and carcinomas with concomitant expression of heparanase, bFGF and VEGF showed the highest MVD. The level of heparanase mRNA expression was directly correlated with the MVD. In addition, heparanase-positive cases had a higher positive ratio of bFGF and VEGF compared with the heparanase-negative cases. These data suggest the possibility that heparanase may contribute to not only cancer cell invasion but also angiogenesis probably through degradation of HS in the ECM and release of bFGF and VEGF from the HS-containing ECM.     

Matrix metalloproteinases-2, -3 and -9 in human term placenta

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that degrade the components of the extracellular matrix (ECM) and are known to be the main mediators of human placentation and parturition. Although there are many studies on the roles and distribution of MMPs in human term placenta, so far none of the studies has investigated the distribution of MMP-2, -3 and -9 in different cells of various placental sites. In this study, we aimed to determine the distribution and enzymatic activities of MMP-2, -3 and -9 with regard to different regions of term human placenta, such as amnion, basal plate, chorionic plate, decidua, chorion laeve, Nitabuch's stria, umbilical cord and placental villi. Eighteen normal human term placentas were obtained after vaginal deliveries. Immunohistochemistry and zymography were performed for MMP-2, -3 and -9 on placental tissue sections and protein extracts, respectively. Nearly all tissues showed immunoreactivity for MMPs. The strongest enzymatic activity for MMP-2 was seen in areas where invasive trophoblast cells invaded maternal tissues. MMP-9 had the highest enzymatic activity at the contact region of fetal and maternal parts, suggesting the importance of MMP-9 in separation of the placenta from the uterine wall during labor. MMP-3 had a similar localization to MMP-9, suggesting that besides gelatinases like MMP-2 and -9, MMP-3 (stromelysin-1) may also have important roles during labor. This study describes the site-specific distribution and activities of MMPs and therefore might help in elucidating the molecular mechanisms in pathologies such as premature rupture of membranes.     

Heparanase expression correlates with invasion and poor prognosis in gastric cancers

Degradation of basement membrane and extracellular matrix structures are important features of the metastatic process of malignant tumors. Human heparanase degrades heparan sulfate proteoglycans, which represent the main components of basement membranes and the extracellular matrix. Because of the role of heparanase in tumor invasion and metastasis, we examined heparanase expression in primary gastric cancers and in cell lines derived from gastric cancers by immunohistochemistry and RT-PCR, respectively. Four of seven gastric cancer cell lines showed heparanase mRNA expression by RT-PCR. Heparanase protein was detected in both the cytoplasm and the nucleus of heparanase mRNA-positive cells by immunohistochemical staining. Heparanase expression was confirmed in 35 (79.5%) of 44 gastric tumor samples by immunohistochemical staining. However, no or weak heparanase expression was detected in normal gastric mucosa. In situ hybridization showed that the mRNA expression pattern of heparanase was similar to that of the protein, suggesting that increased expression of the heparanase protein at the invasive front was caused by an increase of heparanase mRNA in tumor cells. Analysis of the clinicopathologic features showed stronger heparanase expression in cases of huge growing tumors, extensive invasion to lymph vessels, and regional lymph node metastasis. In gastric cancer, patients with heparanase expression showed significantly poorer prognosis than those without such expression (p = 0.006). In conclusion, our findings suggest that high expression of heparanase in gastric cancer is a strong predictor of poor survival.     

Vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor-4 and -10 stimulate trophoblast plasminogen activator system and metalloproteinase-9

Trophoblast invasion, accompanied by degradation of extracellular matrix, is crucial to normal pregnancy development, whereas shallow placental invasion and implantation likely plays a role in the subsequent development of pre-eclampsia. The growth factors vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF) are placental growth factors that activate degradation of extracellular matrix. We determined the effect of VEGF, EGF, FGF-2, FGF-4 and FGF-10 on the plasminogen activator system of first trimester cytotrophoblasts cultured in vitro. We studied the activity of urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor-1 (PAI-1), and 92 kDa gelatinase-B (matrix metalloproteinase-9, MMP-9), using protein gel and reversed gel zymography. The expression pattern of FGF-4 and FGF-10 in human placental sections was determined by immunohistochemistry. FGF-4 was expressed in first trimester villi stroma, primarily in endothelial cells. FGF-10 expression was localized to first trimester extravillous trophoblasts. VEGF, EGF, FGF-4 and FGF-10, but not FGF-2, stimulate the activity of trophoblast uPA, PAI-1 and MMP-9. These results support the hypothesis that specific growth factors modulate the invasive potential of trophoblasts, and therefore may play an important role in early placental development. Our findings may contribute to the understanding of the pathophysiology of diseases associated with shallow placentation, such as pre-eclampsia.     

Heparanase overexpression reduces carrageenan-induced mechanical and cold hypersensitivity in mice

Heparanase controls the structure and functions of extracellular matrix (ECM) by degrading heparan sulfate proteoglycans. Heparanase is involved in inflammatory process through modulating the functions of inflammatory cytokines. The present study aimed to find out whether overexpression of heparanase in mice affects carrageenan-induced localized inflammation and inflammatory hyperalgesia. Without challenge, the heparanase overexpression did not significantly affect the mice in response to mechanical, cold and heat stimulation. Unilateral subcutaneous administration of carrageenan produced hypersensitivity to mechanical and cold in both wildtype and the heparanase overexpression (Hpa-tg) mice 24h after treatment. In comparison to wildtype animals, the Hpa-tg mice showed significantly reduced mechanical and cold hypersensitivity. This may, at least partially, due to the reduced mast cell infiltration at the site of inflammation in Hpa-tg mice. These data support a role for heparanase that reduces localized inflammation and inflammatory hyperalgesia in mice.     

Differentiation of the chorionic plate of the placenta: Cellular and extracellular matrix changes during development in the macaque

Background: The chorionic plate forms the fetal side of the placental disc, and its proper growth and development is important to the formation of a normal placenta. The development and structure of the chorionic plate has received little attention. Therefore, we have conducted a developmental and immunohistochemical study of the chorionic plate of the macaque placenta. Methods: Conventional light and transmission electron microscopy techniques were used to study macaque placental tissues collected from 22 days of gestation to near term. Standard immunoperoxidase methods were used to identify type IV collagen, laminin, and fibronectin in paraffin sections. Results: Early in gestation the chorionic plate trophoblast consisted of an outer layer of syncytiotrophoblast and a single underlying layer of cytotrophoblast. Beginning at about 100 days of gestation, the cytotrophoblast layer became stratified. The cytotrophoblast cells also became surrounded by variable amounts of extracellular matrix containing type IV collagen, laminin, and fibronectin. Ultrastructurally, the matrix contained abundant 10–12 nm diameter microfibrils. During later gestation the syncytiotrophoblast had a tendency to separate from the cytotrophoblast. Conclusions: The chorionic plate of the macaque placenta undergoes several distinctive morphological changes over the course of gestation. During the period of rapid diametrical growth of the disc, the chorionic plate trophoblast consists of a layer of syncytiotrophoblast and a single layer of cytotrophoblast. During later gestation the cytotrophoblast layer stratifies at a time coincident with that at which diametrical growth of the disc slows. The cytotrophoblast cells of later gestation appear synthetically active and at least some of their products are extracellular matrix components that encapsulate many of these cells. These components include type IV collagen, laminin, fibronectin, and microfibrils. © 1994 Wiley-Liss, Inc.     

Galectin-1 and -3 in cells of the first trimester placental bed

The beta-galactoside-binding proteins galectin-1 and -3 are thought to modulate cell-extracellular matrix interactions in cell adhesion and migration. In this study, their occurrence in human trophoblast has been investigated. In the first trimester placenta galectin-1 is expressed in the cytotrophoblast of the mid and distal cell columns, but absent from the villous and proximal column cytotrophoblast. The villous syncytiotrophoblast was also positive. Galectin-3, on the other hand, was uniformly localized in the villous cytotrophoblast and mid and distal cell columns. Immunolocalization of these proteins in placental bed tissue has shown that galectin-1 and -3 are not present in cytokeratin-positive interstitially migrating cytotrophoblast. The co-localization of galectin-1 with extracellular laminin in cultures of cytotrophoblast, choriocarcinoma or decidual stromal cells is consistent with a role in the organization of extracellular matrix and the regulation of cell motility.      

Eosinophil major basic protein: first identified natural heparanase-inhibiting protein

BACKGROUND: Heparanase and eosinophils are involved in several diseases, including inflammation, cancer, and angiogenesis. OBJECTIVE: We sought to determine whether eosinophils produce active heparanase. METHODS: Human peripheral blood eosinophils were isolated by immunoselection and tested for heparanase protein (immunocytochemistry, Western blot), mRNA (RT-PCR) and activity (Na(2)[(35)S]O(4)-labeled extracellular matrix degradation) before and after activation. Heparanase intracellular localization (confocal laser microscopy) and ability to bind to eosinophil major basic protein (MBP) were also evaluated (immunoprecipitation). A model of allergic peritonitis resulting in eosinophilia was induced in TNF knockout and wild-type mice for in vivo studies. RESULTS: Eosinophils synthesized heparanase mRNA and contained heparanase in the active (50-kd) and latent (65-kd) forms. Heparanase partially co-localized with and was bound to MBP. No heparanase enzymatic activity was detected in eosinophils resting or activated with various agonists, including GM-CSF/C5a. Eosinophil lysates and MBP inhibited recombinant heparanase activity in a concentration-dependent manner (100%, 2 x 10(-7) mol/L). Eosinophil peroxidase and eosinophil cationic protein, but not myelin basic protein or compound 48/80, partially inhibited heparanase activity. Poly-l-arginine at very high concentrations caused an almost complete inhibition. In allergic peritonitis, heparanase activity in the peritoneal fluid inversely correlated with eosinophil number. CONCLUSIONS: MBP is the first identified natural heparanase-inhibiting protein. Its presence in the eosinophil granules might indicate a protective function of these cells in diseases associated with inflammation and cancer progression.