WO2008116887A2 - Utilisation de mastocytes péritonéaux comme source d'héparine - Google Patents

Utilisation de mastocytes péritonéaux comme source d'héparine Download PDF

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WO2008116887A2
WO2008116887A2 PCT/EP2008/053580 EP2008053580W WO2008116887A2 WO 2008116887 A2 WO2008116887 A2 WO 2008116887A2 EP 2008053580 W EP2008053580 W EP 2008053580W WO 2008116887 A2 WO2008116887 A2 WO 2008116887A2
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heparin
cells
mast cells
peritoneal
pcmc
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PCT/EP2008/053580
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WO2008116887A3 (fr
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Odile Malbec
Marc Daeron
Michel Arock
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Institut Pasteur
Institut National De La Sante Et De La Recherche Medicale (Inserm)
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Publication of WO2008116887A2 publication Critical patent/WO2008116887A2/fr
Publication of WO2008116887A3 publication Critical patent/WO2008116887A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to peritoneal cell-derived mast cells that are able to respond to IgG antibodies. Further, the present invention relates to cell-models using the peritoneal cell-derived mast cells that respond to IgG stimuli.
  • the cell-models of the present invention are applicable to dermatology, immunology, neurology and rheumatology. In a preferred embodiment, the cell-models of the present invention find utility as a model of multiple sclerosis and rheumatoid arthritis.
  • the present invention also relates to the capability of peritoneal cell-derived mast cells, obtained and cultured as described herein, to release molecules with proteolytic activity and to produce heparin. Accordingly, the present invention provides a new source of heparin.
  • the heparin producing-cells are used as a part of treatment regimens for treating cardiovascular and/or hematological diseases. Further, the heparin producing-cells (or heparin by itself) may also be used in surgical-related applications.
  • Mast cells represent a minor population in tissues, from which they are not readily purified. Moreover, the biological properties of distinct mast cell populations that reside in different tissues are poorly known. Mast cells are not identical in different tissues, and different mast cells may not secrete the same mediators. Mast cells indeed differentiate and mature in peripheral tissues, into which mast cell-committed bone marrow progenitors migrate and where they receive tissue-specific signals (Gurish and Boyce. 2002. Clin Rev Allergy Immunol 22:107-1 18). Thus, mucosal-type mast cells develop in the mucosa of the gastro-intestinal tract and in the lamina intestinal of the respiratory tract where their differentiation depends on T cell-derived cytokines among which IL-3 is critical
  • a good mast cell model should 1 ) be representative of mature differentiated tissue mast cells, and 2) respond not only to IgE, but also to IgG antibodies.
  • BMMC Bone Marrow-derived Mast Cells
  • BMMC are often considered as an in vitro equivalent of mucosal-type mast cells. They, however, are immature cells whose physiological in vivo equivalent is not known. They can indeed reconstitute not only mucosal-type mast cells, but also serosal-type mast cells, when injected intravenously into mast cell-deficient mice (Wershil and GaIIi. 1994. Adv Exp Med Biol 347:39-54).
  • BMMC may correspond to precursors of the mature tissue mast cells which initiate allergies and inflammatory diseases.
  • BMMC express Fc ⁇ RI, Fc ⁇ RIIIA and Fc ⁇ RIIB (Benhamou et al. 1990. J. Immunol. 144:3071-3077). They release mediators when sensitized by IgE and challenged with antigen, and they have been used extensively for studying Fc ⁇ RI signaling. They, however, do not or hardly respond to IgG immune complexes.
  • BMMC are a suitable model for studying either IgE-induced allergic reactions or IgG-induced mast cell-dependent inflammation.
  • Bone Marrow-derived Mast Cells (BMMC) have been used extensively as a mast cell model. BMMC, however, are immature cells which have no known physiological equivalent in tissues. They do not respond to IgG immune complexes. They are therefore not appropriate for studying the physiopathology of IgE-induced allergies or IgG-induced tissue-specific inflammatory diseases.
  • Peritoneal mast cells are mature serosal-type mast cells that respond vigorously to IgG immune complexes. Resident peritoneal mast cells are a minor population of differentiated cells that cannot be readily purified. They represent less than 5% of cells recovered in peritoneal washings from normal mice. They vigorously degranulate not only when sensitized with IgE and challenged with antigen, but also when challenged with preformed soluble IgG immune complexes (Vaz and Prouvost-Danon. 1969. Progr.
  • Heparin Heparin is a mucopolysaccharide with an average molecular weight ranging from
  • the polymeric chain is composed of repeating disaccharide units of D-glucosamine and uronic acid linked by 1— >4 interglycosidic bond.
  • the uronic acid residue could be either D-glucuronic acid or L-iduronic acid. Few hydroxyl groups on each of these monosaccharide residues may be sulfated giving rise to a polymer with that is highly negatively charged.
  • the average negative charge of individual saccharide residues is about 2.3.
  • At least one 3-D structure of heparin exists corresponding to protein data bank code 1 HPN.
  • Heparin is widely used as an injectable anticoagulant and has the highest negative charge density of any known biological molecule. It also may be used to form an inner anticoagulant surface on various experimental and medical devices such as test tubes and renal dialysis machines.
  • heparin used is isolated from pig intestinal mucosa, from where it is extracted by proteolysis, followed by purification on anion exchange resin (for a review on the various methods for preparing heparin, cf. Duclos, "L'heparine: fabrication, structure, proprietes, analyse", Ed. Masson, Paris, 1984).
  • the heparin content in the mucosa-containing aqueous medium is very low and consequently large amounts of mucosa tissue have to be processed.
  • WO 03/035886 describes an alternative method for producing heparin, wherein heparin is obtained from mast cells derived from porcine foetal liver.
  • mast cells are immature. Immature mast cells comprise fewer granules, and therefore less heparin, than mature mast cells.
  • peritoneal mast cells can be expanded in culture to generate large numbers of homogenous cells. At least 1 x 10 8 mast cells could be recovered by culturing the peritoneal cells harvested from two mice for one month.
  • PCMC Peritoneal Cell-derived Mast Cells
  • these PCMCs can therefore be used as a mast cell model that is representative of mature differentiated tissue mast cells.
  • the PCMCs respond both to IgE and to IgG antibodies and this property is due to mild SHIP1-dependent negative regulation.
  • the PCMCs contain and release massive amounts of preformed vasoactive granular mediators and proteases, but secrete no or small amounts of newly formed pro-inflammatory molecules, including ecosanoids, chemokines and cytokines.
  • the granules of cultured PCMC contain massive amounts of heparin. These PCMCs can therefore advantageously be used for producing heparin.
  • peritoneal mast cells can be expanded in vitro by culturing cells in the presence SCF. While BMMCs are obtained by culturing bone-marrow mast cells in the presence of IL-3, it has been unexpectedly been found that peritoneal mast cells can be cultured in the absence of IL-3. On the other hand, peritoneal mast cells can be cultured in the presence of SCF only, which is not the case for bone-marrow mast cells. More specifically, the experimental data shown in the examples demonstrates that like peritoneal mast cells, PCMC respond to IgG antibodies. IgG immune complex- induced responses depended on Fc ⁇ RIIIA and were negatively regulated by Fc ⁇ RIIB.
  • PCMC also respond to IgE antibodies. IgE-induced PCMC responses, however, differed quantitatively and qualitatively from BMMC responses. PCMC secreted no or much lower amounts of newly formed lipid mediators, chemokines and cytokines, but they contain and, upon stimulation, they release much higher amounts of preformed granular mediators. PCMC, but not BMMC, also contained and released upon degranulation molecules with a potent proteolytic activity.
  • PCMC a useful new in vitro model for understanding the physiopathology of mast cells in IgE- and IgG-dependent tissue inflammation.
  • the present invention provides peritoneal mast cells, obtained and cultured by the method described herein and in particular in the examples. As shown in Example 2, these cells contain large amounts of heparin. Such cells thus represent a new source of heparin. The produced cells find use both as mast cell models and in production of heparin.
  • peritoneal cells are seeded and cultured in SCF-containing medium.
  • seeding is at 1x10 6 /ml in complete Opti-MEM supplemented with 4% supernatant of CHO transfectants secreting murine SCF.
  • non-adherent cells are removed and fresh culture medium added to adherent cells.
  • three days later (could be 2-4 days), non-adherent cells and adherent cells recovered with trypsin-EDTA, harvested, pelleted and resuspended in fresh culture medium at a concentration of 3 x 10 5 /ml. The same procedure is repeated twice a week.
  • the length of the culture and the number of repeats of the foregoing process can be determined by monitoring the amount of heparin produced. In a preferred embodiment, culturing is conducted for at least two weeks. In a more preferred embodiment, culturing is conducted for at least one month.
  • peritoneal cells consisting of Fc ⁇ RI + , Kit + , CD19 " , GR1 " , Mad " homogenous cells. This phenotype is characteristic of mast cells. About 1 x 10 8 mast cells could be recovered, after one month, from cultures seeded with the peritoneal cells from two mice. Large numbers of pure mast cells can therefore be generated by culturing mouse peritoneal cells with SCF. These cells contained granules stained with alcian blue/safranin. PCMC granules were much more intensely colored in red than BMMC granules (Fig. 1A), indicating a higher heparin content.
  • heparin may be recovered from the PCMCs by standard isolation and/or purification techniques that have been heretofore utilized for recovering heparin from other sources.
  • the invention is directed to a method of producing a cell producing heparin comprising culturing peritoneal mast cells in the presence of Stem Cell Factor (SCF), in particular fibroblast-derived SCF.
  • SCF Stem Cell Factor
  • Such a culture is preferably performed for a time and under conditions suitable for producing heparin. Such conditions are further detailed below, and may be modified and/or adapted by the skilled in the art.
  • fibroblast-derived Stem Cell Factor is used interchangeably with the terms “Stem Cell Factor” and "SCF” within this specification. These terms refer to the natural kit ligand (see e.g. GaIIi et al. 1994. Adv. Immunology 55:1-96). SCF may be of any origin, e.g. of mouse, human or porcine origin. The term “fibroblast-derived” may refer in particular to a SCF isolated from fibroblasts. However, SCF could be isolated from any other cell type since the sequence of the SCF protein is the same within a given mammal (e.g. human, proc or mouse) irrespective of the cell expressing SCF. The sequence of SCF is well-known in the art (see e.g. Swiss-Prot entries P05532, P10721 and Q2HWD6 for SCF of mouse, human and porcine origin respectively). Preferably, SCF is derived from the same organism as the peritoneal mast cells that are cultured in vitro.
  • the SCF to be added to the cell culture may be produced by any method known in the art. It may for example be obtained from the supernatant of recombinant cells producing SCF such as CHO-KL cells (PIo et al. 2001 Oncogene. 20(46):6752-63), or from a supplier such as R&D (Abigton, UK). In a preferred embodiment, SCF is produced by CHO transfectants secreting murine, porcine or human SCF. In other terms, the SCF may be added to the culture medium by addition of the supernatant of SCF-secreting recombinant cells (e.g. CHO-KL).
  • SCF is produced by CHO transfectants secreting murine, porcine or human SCF.
  • the SCF may be added to the culture medium by addition of the supernatant of SCF-secreting recombinant cells (e.g. CHO-KL).
  • the peritoneal mast cells may be cultured e.g. in the presence of about 10-1000, 20-800, 30-500, 40-400, 50-300, 60-200, 70-100 or 75-90 ng/ml of SCF.
  • CHO-KL cells are seeded at 1 x 10 5 /ml in RPMI medium supplemented with 10% FCS + penicillin/streptomycin and cultured for 4 days. Cell-free supernatant is harvested and added to peritoneal cell cultures. As assessed by ELISA, the conditioned medium thus obtained contained about 80 ng/ml of SCF.
  • the peritoneal mast cells may be of any origin.
  • the peritoneal mast cells are murine, porcine or human peritoneal mast cells.
  • the peritoneal mast cells are most preferably obtained from a mouse.
  • PCMC peripheral cell-derived mast cell
  • peripheral mast cell refers to a cell that is isolated from an organism, but that has not been cultured. The cells may be cultured for instance for at least 2 weeks, at least one month or at least two months.
  • Preferred culture conditions suitable for producing heparin are detailed in the examples.
  • the method of culturing comprises the steps of: a) providing peritoneal cells; b) seeding said peritoneal cells in a liquid culture medium supplemented with a supernatant obtained from a culture of CHO transfectants secreting murine SCF; c) twelve to thirty-six hours later, removing non-adherent cells and adding fresh liquid culture medium to adherent cells; d) two to four days later, recovering non-adherent and adherent cells, and harvesting, pelleting and resuspending said non-adherent and adherent cells in fresh liquid culture medium; and e) repeating step (d) twice a week.
  • Step (a) may be carried out e.g.
  • Step (b) may be carried out by seeding e.g. 1x10 6 cells/ml in a complete liquid culture medium such as Opti-MEM.
  • the liquid culture medium may be supplemented with 1-10%, 3-6% or preferably 4% supernatant of CHO transfectants secreting SCF, in particular murine, porcine or human SCF.
  • the final concentration of SCF in the culture medium may be e.g. of 10-1000, 20-800, 30-500, 40-400, 50-300, 60-200, 70-100 or 75- 90 ng/ml of SCF.
  • the cells may be recovered e.g.
  • fresh medium refers to a liquid culture medium in which cells have not yet been cultivated. Such conditions are suitable for recovering about 1 x 10 8 mast cells after one month of culture. However, it is within the knowledge of the skilled in the art to slightly modify these conditions without impairing the number of cells recovered and/or the amount of heparin produced by the cells.
  • the heparin-producing cell is further immortalized in order to facilitate its cultivation.
  • Such immortalized cells do not constitute a suitable mast cell model, but may be useful in the frame of industrial heparin production.
  • the cell may be immortalized using any method well-known in the art such as viral transformation using Epstein-Barr virus (EBV), Simian virus 40 (SV40) T antigen, adenovirus E1A and E1 B, or human papillomavirus (HPV) E6 and E7, or through expression of the telomerase reverse transcriptase protein (TERT). While such an immortalized cell may lose some of the properties of the primary cell, the skilled in the art can easily verify that it retains the capacity to produce massive amounts of heparin using e.g. staining with alcian blue/safranin.
  • EBV Epstein-Barr virus
  • SV40 Simian virus 40
  • HPV human papillomavirus
  • TERT telomerase reverse transcriptase protein
  • the present invention provides non-transformed peripheral mast cells that are able to respond to IgG stimuli. These cells find application as cell-models in dermatology, immunology, neurology and rheumatology, mainly in the field of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis.
  • Peritoneal Cell-derived Mast Cells are mature serosal-type mouse mast cells which retain most morphological, phenotypic and functional features of peritoneal mast cells. Like peritoneal mast cells, PCMC respond to IgG antibodies. IgG immune complex-induced responses depended on Fc ⁇ RIIIA and were negatively regulated by Fc ⁇ RIIB.
  • PCMC also respond to IgE antibodies. IgE-induced PCMC responses, however, differed quantitatively and qualitatively from BMMC responses. PCMC secreted no or much lower amounts of lipid mediators, chemokines and cytokines, but they contained and released much higher amounts of preformed granular mediators. PCMC, but not BMMC, also contained and released upon degranulation molecules with a potent proteolytic activity. These properties make PCMC a useful new model for understanding the physiopathology of mast cells in IgE- and IgG-dependent tissue inflammation.
  • mast cells are members of the innate immune system, but because they express Fc receptors (FcRs), they can be engaged in adaptive immunity by antibodies.
  • Mast cell FcRs include immunoglobulin E (IgE) and IgG receptors and, among these, activating and inhibitory receptors.
  • IgE immunoglobulin E
  • IgG receptors IgG receptors
  • the engagement of mast cell IgG receptors by immune complexes may or may not trigger cell activation, depending on the type of mast cell.
  • the coengagement of IgG and IgE receptors results in inhibition of mast cell activation.
  • the Src homology-2 domain-containing inositol 5-phosphatase-1 is a major effector of negative regulation. Biological responses of mast cells depend on the balance between positive and negative signals that are generated in FcR complexes.
  • mast cell IgG receptors The exact contribution of human mast cell IgG receptors in allergies remains to be clarified. Increasing evidence indicates that mast cells play critical roles in IgG-dependent tissue-specific autoimmune diseases. Convincing evidence was obtained in murine models of multiple sclerosis, rheumatoid arthritis, bullous pemphigoid, and glomerulonephritis. In these models, the intensity of lesions depended on the relative engagement of activating and inhibitory IgG receptors. In vitro models of mature tissue- specific murine mast cells are needed to investigate the roles of mast cells in these diseases. One such model may allow unraveling unique differentiation/maturation- dependent biological responses of serosal-type mast cells.
  • PCMCs One distinctive property of PCMCs is their ability to respond to stimulation by IgG antibodies. IgG immune complex-induced PCMC activation depended on Fc ⁇ RIIIA, and, as expected, it was negatively regulated by Fc ⁇ RIIB. Surprisingly, the differential responses of PCMCs and BMMCs to IgG immune complexes could not be accounted for by a different ratio of activating/inhibitory FCYRS. Fc ⁇ RIIIA-dependent cell activation was as efficient in both cells, but Fc ⁇ RIIB-dependent negative regulation was more efficient in BMMCs than in PCMCs, revealing that previously unsuspected mechanisms may control Fc ⁇ RIIB-dependent negative regulation.
  • PCMCs produced much lower amounts of lipid mediators during the first half hour of stimulation and no macrophage inflammatory protein-1 ⁇ during the first hours. PCMCs also secreted much less TNF- ⁇ and synthesized fewer cytokines than BMMCs.
  • PCMCs may be informative in studies on the role of serosal-type mature mast cells in tissue inflammation.
  • Mast cells that reside in tissues involved in allergies and inflammatory diseases are indeed of the serosal type, and skin mast cells and synovial mast cells play critical roles in IgE-dependent skin allergies and in IgG- induced arthritis, respectively.
  • the massive amounts of vasoactive mediators and proteases released by serosal-type mast cells within minutes should greatly facilitate the constitution of a local inflammatory infiltrate. Histamine was shown to mediate IgG immune complex-induced, Fc ⁇ RIIIA-dependent inflammation in K/BxN serum-induced arthritis.
  • PA-2 protease-activated receptor-2
  • PAR-2 activation is involved in the control of blood pressure and plasma extravasation, in neutrophil infiltration and proliferation, in the induction of pain, and by stimulating the phagocytosis of melanosomes by keratinocytes, in the control of skin pigmentation.
  • PAR-2 also induces keratinocytes to proliferate and to secrete cytokines.
  • PAR-2 is upregulated in asthma and rheumatoid arthritis.
  • the present invention provides a role for PCMCs as a model for studying the role of serosal-type mature mast cells in tissue inflammation arising from allergy and/or inflammatory diseases.
  • the present invention provides a role for PCMCs as a model for studying the role of serosal-type mature mast cells in multiple sclerosis and rheumatoid arthritis.
  • the present invention provides a model system to study the physiopathology of inflammatory pathologies induced by IgE and IgG.
  • the invention therefore provides a method of preparing an in vitro model system to study the role of serosal-type mature mast cells in tissue inflammation arising from allergy and/or inflammatory diseases comprising preparing a culture of peritoneal cell-derived mast cells by culturing peritoneal cells with SCF, and activating said peritoneal cell- derived mast cells with an IgG or an IgE antibody.
  • the peritoneal cells are cultured with SCF for a time and under conditions suitable to expand peritoneal cell-derived mast cells, for example according to any one of the methods described in the above paragraph entitled "Cells producing heparin".
  • the anti-inflammatory effect of candidate therapeutic drugs can be assessed by monitoring the effect of IgE and/or IgG, preferably
  • This model system finds an application as a method for drug evaluations together or before clinical assays. For example, it may be used in screening assays for identifying anti-inflammatory compounds and/or in functional cell-based assays for characterizing a potential drug during preclinical trials
  • Such a model system may for example be based on a method of identifying the anti-inflammatory effect of a candidate therapeutic drug comprising the steps of: a) preparing a culture of peritoneal cell-derived mast cells by culturing peritoneal cells with SCF; b) splitting the culture of peritoneal cell-derived mast cells into two subcultures; c) activating said peritoneal cell-derived mast cells in one sub-culture with an IgG or an IgE antibody in the presence of said candidate therapeutic drug; d) activating said peritoneal cell-derived mast cells in the other sub-culture with an IgG or an IgE antibody in the absence of said candidate therapeutic drug; and e) comparing the effect of IgG or IgE activation of the peritoneal cell- derived mast cells in the presence of said candidate therapeutic drug as to the effect of activation of the cell-derived peritoneal mast cells in the absence of said candidate therapeutic drug.
  • a reduced activation of the peritoneal cell-derived mast cells in the presence of said candidate therapeutic drug as compared to the activation of the peritoneal cell-derived mast cells in the presence of said candidate therapeutic drug indicates that said candidate therapeutic drug has an anti-inflammatory effect.
  • the effect of IgG or IgE activation of the peritoneal cell-derived mast cells may be assessed by any method well-known in the art. It may for example be assessed by measuring ⁇ -hexamidase release, histamine release, LTC4 production, MIP-1 ⁇ secretion or TNF- ⁇ secretion as described in Example 1.
  • the peritoneal cell-derived mast cells may be activated with an IgG or an IgE antibody. Since the ability to be activated by an IgG antibody is a characteristic feature of PCMCs as compared to BMMCs, the mast cells are preferably activated with an IgG antibody. Most preferably, the antibody is an IgGI , an lgG2a or an lgG2b antibody.
  • the peritoneal cells are cultured with SCF for a time and under conditions suitable to expand peritoneal cell-derived mast cells, for example according to any one of the methods described in the above paragraph entitled "Cells producing heparin".
  • the peritoneal cell-derived mast cells for use as mast cell models are preferably of murine origin.
  • PCMCs prepared as described hereabove contain massive amounts of heparin. More specifically, upon staining with alcian blue/safranin, PCMC granules were much more intensely colored in red than BMMC granules (see Fig. 1A), indicating a higher heparin content. When examined by electron microscopy, more numerous granules were seen in PCMC. They were larger, more homogenous, and had a higher density than BMMC granules (see Fig. 1 B).
  • PCMC contained about 8-fold more ⁇ -hexosaminidase and about 100 to 400-fold more histamine than BMMC (Fig. 2, left panel).
  • BMMC Fig. 2, left panel
  • BMMC (Fig. 2, right panel). Histamine being linked to the granules, it is likely that it reflects the important granule content of PCMCs. It is therefore likely that PCMC contains about 100 to 400-fold more heparin than BMMC.
  • PCMC contain more numerous granules, which have higher granule content, than BCMC. Therefore, the PCMC prepared as described herein can advantageously be used for producing heparin.
  • the invention is thus directed to a method of producing heparin comprising producing a heparin-producing cell according to any of the methods described in the above paragraph entitled "Cells producing heparin", and recovering said heparin from the culture containing said heparin.
  • This method may further comprise the step of purifying said heparin.
  • the invention is also directed to a method for producing a low molecular weight heparin comprising producing heparin by the above method of producing heparin and chemical or enzymatic depolymerizating said heparin to produce said low molecular weight heparin having a weight-average molecular weight ranging from 1 ,000 to 10,000 daltons.
  • the heparin may be recovered from the culture and/or purified by any method well-known in the art.
  • the cells can be harvested and separated from the culture medium, generally by centrifugation or filtration.
  • centrifugation systems can be used.
  • the separation may be carried out by tangential microfiltration using membranes the porosity of which is less than the average diameter of the cells (5 to 20 ⁇ m) while at the same time allowing the other compounds in solution/suspension to pass through.
  • the rate of tangential flow and the pressure applied to the membrane will be chosen so as to generate little shear force (Reynolds number less than 5 000 sec. sup. -1 ) in order to reduce clogging of the membranes and to preserve the integrity of the cells during the separating operation.
  • Various membranes can be used, for example spiral membranes (AMICON, MILLIPORE), flat membranes or hollow fibers (AMICON, MILLIPORE, SARTORIUS, PALL, GF). It is also possible to choose membranes the porosity, the charge or the grafting of which makes it possible to perform a separation and a first purification with respect to possible contaminants which may be present in the culture medium, such as cell proteins, DNA, viruses, or other macromolecules.
  • the heparin is preferably recovered from the culture by a method which keeps the mast cells granules, which contain heparin, intact. This can for example be made by adding water, thereby inducing an osmotic shock.
  • the intact heparin-containing granules can then be separated from the sample by any method well-known in the art. The methods described e.g. in Kr ⁇ ger et al. (Experimental Cell Research Volume 129, Issue 1 , September 1980, pages 83-93) and in Lagunoff and Rickard (American Journal of Pathology, Vol. 154, No. 5, May 1999) may for example be used.
  • the heparin can be harvested from the culture medium after lysis or degranulation of the cells.
  • the heparin has been released from the intracellular content, by degranulation or lysis of all or some of the mast cells, and is present in the culture medium at the time of the separation step, the use of membranes with a smaller porosity may also be envisaged.
  • the cell separation is combined with a step consisting of ultrafiltration on one or more membranes, the organization and the porosity of which make it possible to concentrate the heparin and to separate it from the other species present in the medium, as a function of the size and the molecular weight and, optionally, of the electrical charge, or of the biological properties.
  • the cutoff threshold of the membranes is preferably between 1000 and 5 kDa.
  • Use may be made of membrane systems similar to those used for microfiltration, for example spiral membranes, flat membranes or hollow fibers.
  • Use may advantageously be made of membranes which make it possible to separate and purify the heparin due to their charge properties or their properties of grafting of ligands exhibiting affinity for heparin (for example antibodies, ATIII, lectin, peptides, nucleotides, etc.).
  • the degranulation may be caused by the binding of specific ligands to the receptors present at the surface of the mast cells, for example IgG or IgE, or by other agents including but not limited to cytotoxic agents, enzymes, polysaccharides, lectins, anaphylatoxins, basic compounds (compound 48/80, substance P, etc.), calcium (A23187 ionophore, ionomycin, etc.).
  • the mast-cell lysis can be induced, for example, by osmotic shock using hypotonic or hypertonic solutions, by thermal shock (freezing/thawing), by mechanical shock (for example sonication or pressure variation), by the action of chemical agents (NaOH, THESIT.
  • TM., NP40.TM., TWEEN 20.TM., BRIJ-58.TM., TRITON X.TM.-100, etc. or by enzyme lysis (papain, trypsin, etc.), or by a combination of two or more of these methods.
  • the cell lysate can be subjected to one or more enzyme digestions (pronase, trypsin, papain, etc.); - the heparin-protein bonds can be hydrolyzed in alkali medium, in the presence of sulfates or chlorides;
  • a treatment in acid medium for example with trichloroacetic acid under cold conditions
  • acid medium for example with trichloroacetic acid under cold conditions
  • an ionic solution which makes it possible to dissociate the GAG-protein interactions
  • an extraction with guanidine after enzyme hydrolysis, can be carried out to purify the solubilized heparin. It is for example possible to precipitate it with potassium acetate, with a quaternary ammonium, or with acetone.
  • These purification steps may optionally be followed or replaced with one or more chromatography steps, in particular an anion exchange chromatography step and/or an affinity chromatography step.
  • the purification may be carried out as described by Volpi (J Chromatogr B Biomed Appl. 1996 Oct 11 ;685(1 ):27-34).
  • Heparin which is a highly sulfated glycosaminoglycan, finds significant utility in treatment regimens for cardiovascular and/or hematological diseases, as well as in surgical applications. Heparin is widely used as an injectable anticoagulant and has the highest negative charge density of any known biological molecule. It also may be used to form an inner anticoagulant surface on various experimental and medical devices such as test tubes and renal dialysis machines. In an aspect of the present invention, the heparin producing-cells (or heparin by itself) are used as a part of treatment regimens for treating cardiovascular and/or hematological diseases (see above).
  • the heparin producing-cells may also be used in surgical-related applications (see above).
  • the heparin may be isolated and/or purified from the cells producing the same and used in the isolated and/or purified form. Additional applications for which heparin finds application, include:
  • Anticoagulant therapy in prophylaxis and treatment of venous thrombosis and its extension e.g., deep-vein thrombosis.
  • Acute coronary syndrome e.g., myocardial infarction.
  • Treatment of interstitial cystitis e.g., myocardial infarction.
  • heparin The key structural unit of heparin is thought to be a unique pentasaccharide sequence (below).
  • This sequence consists of three D-glucosamine and two uronic acid residues.
  • the central D-glucosamine residue contains a unique 3-0-sulfate moiety that is rare outside of this sequence.
  • Four sulfate groups on the D-glucosamines are found to be critical for retaining high anticoagulant activity. Elimination of any one of them results in a dramatic reduction in the anticoagulant activity. Removal of the unique 3-O-sulate group results in complete loss of the anticoagulant activity. Removal of sulfate groups other than the critical ones seems to not affect the anticoagulant activity.
  • Heparin contains a unique five-residue sequence
  • GlcNS(6S) 2-deoxy-2-sulfamido- ⁇ -D-glucopyranosyl-6-O-sulfate which is recognized by and forms a high-affinity complex with antithrombin (e.g., antithrombin III).
  • antithrombin e.g., antithrombin III
  • antithrombin III Upon binding heparin, antithromibin III undergoes a conformational change, which results in its active site being exposed.
  • the activated antithrombin III resulting from the formation of antithrombin - heparin complex becomes a more rapid acting inhibitor of thrombin, factor X, and several other coagulation enzymes (IX, Xl & XII).
  • antithrombin - heparin complex greatly increases the rate of inhibition of two principle procoagulant proteases, factor Xa and thrombin.
  • the normally slow rate of inhibition of factor Xa and thrombin ( ⁇ 10 3 - 10 4 M “ V 1 ) by antithrombin alone is increased about a 1 , 000-fold by heparin (Bjork I, Lindahl U. (1982). "Mechanism of the anticoagulant action of heparin". MoI. Cell. Biochem. 48: 161-182).
  • thrombin III The conformational change in antithrombin III on heparin binding mediates its inhibition of factor Xa.
  • thrombin For thrombin inhibition however, thrombin must also bind to the heparin polymer at a site proximal to the pentasaccharide.
  • the high negative charge density of heparin contributing to its very strong electrostatic interaction with thrombin (Cox, M.; Nelson D. (2004). Lehninger, Principles of Biochemistry. Freeman, 1100).
  • the formation of a ternary complex between antithrombin III, thrombin and heparin results in the inactivation of thrombin.
  • heparin's activity against thrombin is size dependent, the ternary complex requiring at least 18 saccharide units for efficient formation (Petitou M, Herault JP, Bernat A, Driguez PA, et al. (1999). "Synthesis of Thrombin inhibiting Heparin mimetics without side effects". Nature 398: 417-422).
  • anti factor Xa activity only requires the pentasaccharide binding site.
  • heparin acts to prevent the formation of clots and extension of existing clots within the blood.
  • heparin does not break down clots that have already formed (the tissue plasminogen activator will), it allows the body's natural clot lysis mechanisms to work normally to break down clots that have already formed.
  • heparin exits as the anion at physiologic pH and is usually administered as the sodium salt.
  • the present invention is not limited to the sodium salt.
  • Other salt forms, including but not limited to the lithium salt and ammonium salt, may also be used.
  • Heparin may be administered parenterally. Alternatively, heparin may be administered by intravenous injection or subcutaneous (under the skin) injection. Intramuscular injections (into muscle) are generally avoided because of the potential for forming hematomas. However, the present invention also contemplates intramuscular injection as a means of delivery. Topical administration may also be employed.
  • heparin be administered frequently or as a continuous infusion.
  • continuous infusion intravenous administration is mentioned.
  • the dosage of heparin sodium should be adjusted according to the patient's coagulation test results. For example, when heparin sodium is given by continuous intravenous infusion, the coagulation time may be determined approximately every four hours in the early stages of treatment. When the drug is administered intermittently by intravenous injection coagulation tests may be performed before each injection during the early stages of treatment and at appropriate intervals thereafter.
  • APTT is 1.5 to 2 times normal or when the whole blood clotting time is elevated approximately 2.5 to 3 times the control value.
  • tests for adequacy of dosage are best performed on samples drawn four to six hours after the injections.
  • Periodic platelet counts, hematocrits and tests for occult blood in stool are recommended during the entire course of heparin therapy, regardless of the route of administration. Although dosage must be adjusted for the individual patient according to the results of suitable laboratory tests, the dosage schedules of the following table may be used as guidelines.
  • Heparin referred to above relates to that directly obtained from mastocytes.
  • the heparin-producing cells are used directly.
  • the heparin produced by these cells is isolated and/or purified by conventional techniques.
  • Low molecular weight heparins in contrast, consist of only short chains of polysaccharide.
  • Low-molecular-weight heparin is derived from standard heparin through either chemical or enzymatic depolymerization. Whereas standard heparin has a weight- average molecular weight of 5,000 to 40,000 daltons, low-molecular-weight heparin ranges from 1 ,000 to 10,000 daltons, resulting in properties that are distinct from those of traditional heparin.
  • the following methods of production of low-molecular-weight heparin from standard heparin have been used in the art and may be used in the present invention: • Oxidative depolymerisation with hydrogen peroxide.
  • Beta-eliminative cleavage by the heparinase enzyme • Deaminative cleavage with nitrous acid.
  • Low-molecular-weight heparin binds less strongly to protein, has enhanced bioavailability, interacts less with platelets and yields a very predictable dose response, eliminating the need to monitor the aPPT.
  • Low- molecular-weight heparin like standard heparin, binds to antithrombin III; however, low- molecular-weight heparin inhibits thrombin to a lesser degree (and Factor Xa to a greater degree) than standard heparin (Hirsch J, Raschke R, Warkentin TE, Dalen JE, Deykin D, Poller L. Heparin: mechanism of action, pharmacokinetics, dosing considerations, monitoring, efficacy and safety. Chest 1995;108(4 suppl): 258-75).
  • the clinical advantages of low-molecular-weight heparin include predictability, dose-dependent plasma levels, a long half-life and less bleeding for a given antithrombotic effect (Warkentin TE, Levine MN, Hirsch J, Horsewood P, Roberts RS, Gent M, et al. Heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin. N Engl J Med 1995;332:1330-5).
  • immune- mediated thrombocytopenia is not associated with short-term use of low-molecular-weight heparin (Warkentin et al), and the risk of heparin-induced osteoporosis may be lower than the risk with the use of standard heparin.
  • Low-molecular-weight heparin is administered according to body weight once or twice daily. Thus, continued monitoring is not necessary.
  • the low-molecular-weight heparin is administered to a subject in need of such treatment one to six times daily, preferably one or two times daily, with a total daily dosage ranging from 30 to 150 mg per day, preferably from 45 to 125 mg per day.
  • the amount to be administered is calculated as ranging from 0.3 to 2 mg/kg per day, which may be administered in divided dosages. A more preferred amount is calculated as ranging from 0.5 to 1.5 mg/kg per day, and most preferred as 1 mg/kg per day.
  • the invention further contemplates a method of treating or preventing a disorder selected from the group consisting of venous thrombosis and its extension, post-operative deep venous thrombosis and pulmonary embolism in patients undergoing major abdomino-thoracic surgery and/or are at risk of developing thromboembolic disease, pulmonary embolism, atrial fibrillation with embolization, acute and chronic consumptive coagulopathies, clotting in arterial and cardiac surgery, peripheral arterial embolism, acute coronary syndrome, and interstitial cystitis comprising administering heparin or low molecular weight heparin obtained according to the methods of the invention to a subject.
  • an effective amount of heparin and/or of low molecular weight heparin is preferably administered to the subject in need thereof.
  • the subject in need thereof may for example suffer from, or be at risk of suffering from, a disorder selected from the group consisting of venous thrombosis and its extension, post-operative deep venous thrombosis and pulmonary embolism in patients undergoing major abdomino-thoracic surgery and/or are at risk of developing thromboembolic disease, pulmonary embolism, atrial fibrillation with embolization, acute and chronic consumptive coagulopathies, clotting in arterial and cardiac surgery, peripheral arterial embolism, acute coronary syndrome, and interstitial cystitis.
  • venous thrombosis and its extension refers to the disorders described in Lopez et al. (Hematology (2004) pages 439-456).
  • the invention also pertains to heparin and/or low molecular weight heparin obtainable by the methods according to the invention for use in the treatment or prevention of a disorder selected from the group consisting of venous thrombosis and its extension, post-operative deep venous thrombosis and pulmonary embolism in patients undergoing major abdomino-thoracic surgery and/or are at risk of developing thromboembolic disease, pulmonary embolism, atrial fibrillation with embolization, acute and chronic consumptive coagulopathies, clotting in arterial and cardiac surgery, peripheral arterial embolism, acute coronary syndrome, and interstitial cystitis.
  • FIG. 1 Characterization of PCMC.
  • A Morphology of cultured mast cells. PCMCs and BMMCs were cytocentrifuged, stained with alcian blue/safranin and observed under the microscope.
  • B Ultrastructure of cultured mast cells. PCMC and BMMC were observed by electron microscopy.
  • Figure 2 Fc ⁇ RI-dependent ⁇ -hexosaminidase and histamine release.
  • PCMC and BMMC sensitized with mouse IgE anti-DNP were challenged with the indicated concentrations of DNP-BSA for 10 min at 37O.
  • 1 x 1 O 5 cells were used for ⁇ - hexosaminidase release and 1 x 10 6 cells were used for histamine release, ⁇ - hexosaminidase and histamine were measured in supernatants and in cell lysates.
  • Left panels show the mean ⁇ SD of values measured in BMMC and PCMC lysates.
  • Right panels show the relative (percentage) and the absolute amounts of ⁇ -hexosaminidase and histamine released by individual cell populations.
  • the insert shows histamine released by BMMC with an expanded vertical scale.
  • mice C57BL/6 mice, purchased from IFFA-CREDO (Saint-Germain-sur-L'Arbresle, France) or from Charles River Laboratories (L'Arbresle, France), were used as donors of bone marrow and peritoneal cells.
  • BALB/c mice purchased from IFFA-CREDO, were used for immunizations.
  • SHIP1 " ' " mice, generated by Dr. Gerald Krystal (The Terry Fox Laboratories, Vancouver, Canada), and SHIP1 +/+ littermate controls were kindly provided by Dr. Michael Huber (Max Plank lnstitut fur Immunbiologie, Freiburg, Germany). Bone marrow and peritoneal cells from RFc ⁇ llB " ' " , RFc ⁇ lllA " ' " and wt littermate controls mice
  • Femoral bone marrow cells were collected and cultured in Opti-MEM supplemented with 10% Fetal Calf Serum (FCS), 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin (complete Opti-MEM) and 4% supernatant of X63 transfectants secreting murine IL-3. Cultures were passaged every 3 days by resuspending pelleted cell in fresh culture medium at a concentration of 3 x 10 5 /ml. Peritoneal cells were collected from the same mice injected with 2 ml RPMI intraperitoneal ⁇ .
  • the rat anti-mouse Fc ⁇ RIIB/IIIA mAb 2.4G2 was affinity- purified from culture supernatants on Protein G-sepharose (Amersham Little Chalfont, UK).
  • the mouse anti-Ly-17.2 mAb K9.361 Holmes et al. 1985. Proc. Natl. Acad. Sci. USA 82:7706-7710
  • the mouse IgE anti-DNP mAb 2682-I Liu et al. 1980. J. Immunol. 124:2728-2737 were used as culture supernatants.
  • K9.361 which recognizes the Ly-17.2 alloantigen, encoded by the Ly-17b allele of the fcgr2b gene, was demonstrated as being an Fc ⁇ RI IB-specific mAb with no cross-reactivities to other Fc ⁇ Rs, including Fc ⁇ RIIIA (Schiller et al. Eur J Immunol 30:481-490).
  • the IgE concentration in 2682-I supernatant was 10 ⁇ g/ml as titrated by ELISA.
  • Allophycocyanin (APC)-labeled anti CD117 antibodies, Phycoerythrin (PE)-labeled anti-CD19 antibodies, PE-labeled anti-GR1 antibodies and PE-labeled anti-Mad antibodies were from BD-Pharmingen (Le Pont de Claix, France). Fluorescein (FITC)-labeled anti Fc ⁇ RI antibodies were from e-biosciences (San Diego, CA).
  • FITC-labeled Mouse anti-Rat (MAR) F(ab') 2 fragments FITC-labeled Goat anti- Mouse (GAM) F(ab') 2 fragments
  • FITC-labeled Goat anti-Rabbit (GAR) F(ab') 2 fragments FITC-labeled Goat anti-Rabbit (GAR) F(ab') 2 fragments
  • Rabbit anti-Mouse (RAM) F(ab') 2 fragments and intact IgG antibodies were from Jackson ImmunoResearch (Westgrove, PA).
  • Bovine Serum Albumin (BSA) from Sigma Aldrich (Lyon, France), was dinitrophenylated with dinitrobenzene-sulfonic acid (Eastman Kodak, Rochester, NY). DNP 15 -BSA was obtained.
  • GST Mouse anti-Glutathione S-Transferase serum was raised in BALB/c mice injected once with purified GST in complete Freund's adjuvant and twice in incomplete Freund's adjuvant intraperitoneal ⁇ . IgG were affinity- purified from serum on Protein G-sepharose.
  • Phycoerythrin (PE)-labeled anti-IL-6 and anti-IL-10 antibodies, and biotinylated anti-TGF ⁇ 1 antibodies were from Beckton Dickinson (Franklin Lakes, NJ).
  • PE-labeled anti-IFN ⁇ , anti-TNF- ⁇ , and anti-IL-4 antibodies were from Serotec (Cergy Saint-Christophe, France).
  • Biotinylated anti-IL-13 antibodies were from R&D Systems (Lille, France). FITC-labeled streptavidine was from Molecular Probes (Carlsbad, CA). The mouse anti-FcRD mAb JRK was the one described in Rivera et al. (Molecular Immunology, formerly known as Immunochemistry, Volume 25, Issue 7, Pages 647-661. July 1988)). Rabbit anti-Lyn, anti-SHP-1 , anti-SHP-2, anti-Gab2, anti-Sos and anti-PLC- ⁇ 1 antibodies were from Upstate biotechnology (Waltham, MA), as well as mouse anti-Vav antibodies.
  • Rabbit anti-Grb2, anti-PLC- ⁇ 2 and anti-SHIP1 were from Santa Cruz (Santa Cruz, CA).
  • Mouse anti-Fyn antibodies were from Transduction laboratories (Lexington, KY).
  • Rabbit anti-Erk and anti-Akt antibodies were from Cell signaling (Beverly, MA).
  • Horse Radish Peroxidase (HRP)-labeled Goat anti-Rabbit (GAR) and Goat anti-Mouse (GAM) were from Santa Cruz. Direct immunofluorescence. Cells were incubated for 5 min at OO with 10 ⁇ g/ml
  • Electron microscopy Cells were fixed with 2.5% glutaraldehyde in 0.01 M PBS pH 7.4 for 1 hr at 4O, post-fixed with 2% osmium tetr oxyde for 1 hr, dehydrated with ethanol, and embedded in Epon epoxy resin. Ultra-thin sections (80-100 nm) were stained with uranyl acetate and lead citrate, and examined at 80 kV using a JEOL (JEM-1005) electron microscope.
  • Mast cells identified by their morphology under the microscope, were individually picked-up from peritoneal cells, resuspended at 37O in soft agar dissolved in SCF-containing maxim m and layered over medium- containing soft agar previously layered over adherent SCF-secreting CHO transfectants. ⁇ -hexosaminidase release. Mast cells, sensitized with IgE anti-DNP, were challenged for 10 min at 37O with indicated reagen ts.
  • Non sensitized mast cells were challenged for 10 min at 37°C with preformed immune complexes made by incubating serum anti-GST or affinity-purified IgG anti-GST with GST at the indicated dilutions or concentrations for 15 min at 37O immediately befor e use. Reactions were stopped on ice. Five ⁇ l supernatant were mixed with 45 ⁇ l ⁇ -hexosaminidase substrate (Sigma Aldrich) and incubated for 2 hr at 37O. 0.2 M glyc ine pH 10 was added, and absorbance at 405 nm was measured.
  • LTC4 production Mast cells, sensitized with IgE anti-DNP, were challenged with DNP-BSA for 20 min at 37O. LTC4 was titrated in su pernatants by competitive ELISA (Neogen corporation, Lexington, KY). MIP-1 ⁇ secretion. Mast cells, sensitized with IgE anti-DNP, were challenged with
  • DNP-BSA for indicated periods of time at 37O.
  • MIP- 1 ⁇ was titrated in supernatants by ELISA (R&D Systems, Lille, France).
  • TNF-a secretion Mast cells, sensitized with IgE anti-DNP, were challenged for various periods of time at 37O with DNP-BSA.
  • TNF- ⁇ was titrated in supernatants by a cytotoxicity assay on L929 cells as described (Latour et al. 1992. J. Immunol. 149:2155- 2162).
  • Morphological assay for mast cell degranulation Cultured mast cells or peritoneal cells, sensitized with mouse IgE, were challenged for 5 min at 37O with RAM F(ab') 2 - Reactions were stopped on ice, and cells were stained with Toluidine Blue.
  • cells were lysed by being boiled for 5 min at 95O in 10 mM Tris pH 7.4 containing 1 % SDS. Lysates were passaged 6 times through a gauge-26 needle, centrifuged at 12,000 rpm for 10 min at 4 O and immediately electrophores ed.
  • Protease activity secretion Mast cells, sensitized with IgE anti-DNP, were challenged for the indicated periods of time at 37°C with DNP-BSA. Reactions were stopped on ice. Proteolytic activity was measured in supernatants using an enzymatic assay. Briefly, 100 ⁇ l were incubated for 30 min at 37O with 10 ⁇ l 0.2 M Tris pH 7.8, 0.02 M CaCI 2 and 10 ⁇ l 0.4% resorufin-labeled casein (Roche Dignostic, Penzberg, Germany). 100 ⁇ l 5% trichloroacetic acid were added and plates were incubated for 10 min at 37O before being centrifuged for 5 min. 80 ⁇ l supernatants were mixed with 120 ⁇ l 0.5 M Tris pH 8.8. The absorbance was read at 570 nm.
  • Example 2 High numbers of homogenous mature serosal-type mast cells can be generated in culture from mouse peritoneal cells.
  • Peritoneal cells from two C57BL/6 mice were cultured in SCF-containing medium as described in Materials & Methods.
  • BMMC were generated in parallel from bone marrow cells of the same two mice cultured in IL-3-containing medium.
  • One month-old cultures of both types consisted of Fc ⁇ RI ⁇ Kit + , CD19 " , GR1 " , Mad " homogenous cells.
  • This phenotype is characteristic of mast cells. About 1 x 10 10 and 1 x 10 8 mast cells could be recovered, after one month, from bone marrow and from peritoneal cell cultures, respectively. Large numbers of pure mast cells can therefore be generated by culturing mouse peritoneal cells with SCF. These cells will be referred to as Peritoneal Cell-derived Mast Cells (PCMC).
  • PCMC Peritoneal Cell-derived Mast Cells
  • PCMC and BMMC contained granules stained with alcian blue/safranin.
  • PCMC granules were much more intensely colored in red than BMMC granules (Fig. 1A), indicating a higher heparin content.
  • Fig. 1A When examined by electron microscopy, more numerous granules were seen in PCMC. They were larger, more homogenous, and had a higher density than BMMC granules (Fig. 1 B).
  • These staining and morphological properties are characteristic features of mature mast cells.
  • PCMC and BMMC contained mast cell-specific protease transcripts. Both expressed mMCP-2, mMCP-4, mMCP-5, mMCP-6, mMCP-7 and mMCP-8.
  • BMMC but not PCMC, expressed mMCP-9 and mMCP-10.
  • Example 3 PCMC respond not only to IqE and antigen, but also to IqG immune complexes.
  • BMMC and PCMC expressed comparable levels of Fc ⁇ RI.
  • PCMC and BMMC released similar percentages of ⁇ -hexosaminidase. Inhibition observed in excess of antigen was however more pronounced in BMMC than in PCMC.
  • PCMC When challenged with preformed immune complexes, non-sensitized PCMC dose- dependently released ⁇ -hexosaminidase. Under the same conditions, BMMC did not respond or very poorly. Immune complex-induced ⁇ -hexosaminidase release was enhanced in Fc ⁇ RIIB " ' " PCMC.
  • Fc ⁇ RIIB enabled BMMC to respond to immune complexes and the responses of Fc ⁇ RIIB " ' " BMMC were of the same magnitude as those of Fc ⁇ RIIB " ' " PCMC.
  • ⁇ -hexosaminidase release was abrogated in Fc ⁇ RIIIA " ' " BMMC and PCMC (Fig. 2B, right panel).
  • wt BMMC and PCMC expressed comparable levels of Fc ⁇ R as assessed by immunofluorescence with the anti-Fc ⁇ RIIB+IIIA mAb 2.4G2, and comparable levels of Fc ⁇ RIIB as assessed with the anti-allotypic mAb K9.361.
  • Fc ⁇ RIIB could therefore negatively regulate Fc ⁇ RIIIA-dependent cell activation in both BMMC and PCMC, but no difference in the relative expression of activating and inhibitory receptors could explain why it prevented BMMC, but not PCMC, from responding to IgG immune complexes.
  • Fc ⁇ RI IB-dependent negative regulation its ability to inhibit Fc ⁇ RI-dependent mast cell activation was investigated in the two cell types.
  • BMMC and PCMC sensitized with mouse IgE were challenged either with RAM F(ab') 2 fragments, to aggregate Fc ⁇ RI, or with intact RAM IgG, to co-aggregate Fc ⁇ RI with Fc ⁇ RIIB in wt cells.
  • Fc ⁇ RI IB-dependent inhibition was more pronounced in BMMC than in PCMC.
  • SHIP1 is the main intracellular effector of Fc ⁇ RI IB-dependent negative regulation. It also controls Fc ⁇ RI signaling.
  • BMMC and PCMC were generated from SHIP1 " ' " and from wt littermate controls.
  • the deletion of SHIP1 increased immune complex-induced ⁇ -hexosaminidase release in BMMC but, surprisingly, not in PCMC.
  • the deletion of SHIP1 abrogated RAM IgG-induced Fc ⁇ RI IB-dependent negative regulation of Fc ⁇ RI-dependent ⁇ - hexosaminidase release in both BMMC and PCMC. Inhibition was again more marked in BMMC than in PCMC.
  • BMMC and PCMC indeed contained comparable amounts of SHIP1 transcripts, as assessed by RT-PCR, and comparable amounts of the SHIP1 protein, as assessed by intracellular immunofluorescence.
  • Example 4 PCMC secrete small amounts of newly formed lipid mediators, chemokines and cytokines in response to IqE and antigen.
  • BMMC secreted about 30 times more LTC4 than PCMC, as assessed by ELISA.
  • BMMC secreted MIP-1 ⁇ , as assessed by ELISA.
  • TNF- ⁇ as assessed by a cytotoxicity assay.
  • PCMC secreted much less TNF- ⁇ than BMMC.
  • PCMC containing intracellular TNF- ⁇ were also detected by immunofluorescence following stimulation by PMA + ionomycin for 2 hr. They were less numerous than BMMC containing TNF- ⁇ observed following the same treatment. PMA + ionomycin-treated
  • PCMC and BMMC also contained IL-6, and lower numbers of cells contained, IL-13, but not IL-4, IL-10, TGF- ⁇ 1 or IFN ⁇ .
  • PCMC therefore secreted no or much lower amounts of LTC4, MIP-1 ⁇ and TNF- ⁇ .
  • PCMC could however synthesized the same set of cytokines as BMMC in response to PMA + ionomycin.
  • Example 5 PCMC release large amounts of preformed granular mediators in response to IgE and antigen.
  • PCMC contained about 8-fold more ⁇ -hexosaminidase and about 100- fold more histamine than BMMC (Fig. 2, left panel).
  • PCMC released much higher absolute amounts of ⁇ -hexosaminidase and even higher amounts of histamine than BMMC (Fig. 2, right panel), even though both mast cells released comparable percentages of these mediators (Fig. 2, middle panel).
  • both cells released a higher percentage of histamine than of ⁇ -hexosaminidase, when challenged identically in the same experiment.
  • PCMC but not BMMC, therefore contain a highly efficient protease activity that is released upon cell lysis and hydrolyzes several high-mw intracellular proteins. Proteolysis could however be prevented if PCMC were lysed in SDS-containing buffer and immediately boiled before electrophoresis. Under these conditions, SHIP1 , but also other molecules not seen in PCMC TX100 lysates, such as Akt, were readily detectable, and in similar amounts as in BMMC.
  • proteolytic enzymes contained in PCMC could be released and hydrolyze an exogenous substrate such as casein.
  • Proteolytic activity was indeed detected in supernatants of PCMC sensitized with IgE anti-DNP and challenged with DNP-BSA for 10 min, but not in supernatants of IgE-sensitized, but not challenged PCMC.
  • no proteolytic activity was detected in supernatants from IgE-sensitized BMMC, whether they were or not challenged with antigen.
  • Proteolytic activity was released from IgE-sensitized PCMC with the same kinetics as ⁇ -hexosaminidase, i.e. within the first 10 min after antigen challenge, and it did not increase thereafter. Proteolytic enzymes are therefore likely to be contained in PCMC granules and to be released upon degranulation.
  • PCMCs were characterized, and it was shown that PCMC is a new model of cultured mouse mast cells which markedly differ from BMMC. Indeed, PCMCs consist of mature differentiated mast cells which retain most of the properties of serosal-type peritoneal mast cells. This makes PCMC useful for studying immunopathological processes. Serosal-type mast cells are indeed present in tissues involved in allergies and inflammatory diseases. Skin mast cells and synovial mast cells, for instance, both of the serosal type, play critical roles in skin allergies and in the murine model of IgG-induced autoimmune rheumatoid arthritis recently described in K/BxN mice, respectively. Mast cells can be obtained by fractionation techniques from mouse peritoneal cells
  • mast cells No more than 1 x 10 5 mast cells can however be obtained per mouse, which greatly limits investigations.
  • a few hundred million homogenous mast cells can be readily generated from the peritoneal cells of two mice and kept in culture for at least two months. These cells are typical mast cells as judged by their expression of Fc ⁇ RI and Kit, their morphology, their histamine content and their functional features. They are mature mast cells as judged by their intense staining with alcian blue/safranin, and by the high number and the dense structure of their granules.
  • PCMC PCMC
  • biological responses differ quantitatively and qualitatively, from BMMC responses.
  • Fc ⁇ RI aggregation triggered the same responses in BMMC and PCMC.
  • BMMC and PCMC released comparable percentages of ⁇ -hexosaminidase and comparable percentages of histamine.
  • PCMC contained almost 10-fold higher amounts of ⁇ -hexosaminidase and, like peritoneal mast cells, about 100-fold higher amounts of histamine than BMMC. Consequently, PCMC released much more granular mediators than BMMC within the first minutes of activation via Fc ⁇ RI.
  • PCMC produced much lower amounts of lipid mediators during the first half hour of stimulation, and no MIP-1 ⁇ during the first hours. They also secreted much less TNF- ⁇ than BMMC. Noticeably, no TNF- ⁇ was detected in supernatants of either cell type at 10 min, when degranulation was completed, and no TNF- ⁇ was detected by intracellular immunofluorescence in non stimulated cells, indicating that this cytokine is not stored in BMMC or PCMC granules. TNF- ⁇ , however, became detectable intracellular ⁇ , several hours following stimulation.
  • PCMC Another biological response was unique to PCMC. Indeed, Fc ⁇ RI aggregation triggered a release of proteolytic activity in PCMC, but not in BMMC.
  • the responsible proteases were not identified. They hydrolyzed cleavage sites that are rare in low-mw proteins such as casein, and more frequent in high-mw proteins such as SHIP1. They are present in resting cells since proteolysis was observed in PCMC lysates before stimulation. Noticeably, proteases released in PCMC supernatants did not account for the low amounts of TNF- ⁇ found in these supernatants.
  • PAR-2 is expressed by neutrophils, endothelial cells, vascular smooth muscle cells, neurons and glial cells, enterocytes, keratinocytes and many tumor cells. PAR-2 activation is involved in the control of blood pressure and plasma extravasation, in neutrophil infiltration and proliferation, in the induction of pain and, by stimulating the phagocytosis of melanosomes by keratinocytes, in the control of skin pigmentation. PAR-2 also induces keratinocytes to proliferate and to secrete cytokines. Interestingly, PAR-2 is up-regulated in asthma and rheumatoid arthritis. Lipid mediators account for the late-phase reaction which develops locally.
  • Cytokines and chemokines account for the chronic inflammatory reaction which is responsible for most of the long- lasting clinical symptoms of allergic diseases.
  • TNF- ⁇ which induces bronchial hyperresponsiveness, airway infiltration by neutrophils and eosinophils, activation of airway smooth muscles and myofibroblasts, and which upregulates the expression of adhesion molecules, was recognized as playing a major role in asthma- associated remodeling and pulmonary inflammation, especially in asthma refractory to corticosteroid therapy.
  • TNF- ⁇ also critically contributes to the pathogenesis of rheumatoid arthritis.
  • BMMC are potent secretors of proinflammatory chemokines and cytokines.
  • Physiological BMMC equivalents may exist in the bone marrow and, transiently, in the circulation, but not in peripheral tissues. They therefore cannot account for tissue inflammation. If, as discussed above, PCMC result from an expansion of preexisting peritoneal mast cells and are representative of serosal- type mast cells, these mast cells cannot either account themselves for tissue inflammation. They are indeed poor secretors of cytokines. Other cells, which are known to converge to allergic sites and which infiltrate tissues are required for inflammation to be generated. The massive amounts of vasoactive mediators and proteases that are released by PCMC within minutes should greatly facilitate the subsequent constitution of an inflammatory infiltrate. One may therefore speculate that serosal-type mast cells function as promoters rather than as effectors of inflammation in allergies and autoimmune diseases.

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  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des mastocytes dérivés de cellules péritonéales capables de répondre à des stimuli IgG. Cette invention concerne plus spécifiquement l'aptitude de mastocytes dérivés de cellules péritonéales, développés in vitro par la culture de cellules péritonéales en présence du facteur de croissance des cellules souches (SCF), à produire de l'héparine. Ainsi, cette invention procure une nouvelle source d'héparine. Selon un aspect de cette invention, les cellules productrices d'héparine (ou l'héparine elle-même) sont utilisées dans le cadre de régimes de traitement visant à traiter des maladies cardio-vasculaires et/ou hématologiques. Les cellules productrices d'héparine (ou l'héparine elle-même) peuvent également être utilisées dans des applications associées à la chirurgie.
PCT/EP2008/053580 2007-03-26 2008-03-26 Utilisation de mastocytes péritonéaux comme source d'héparine WO2008116887A2 (fr)

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US60/908,040 2007-03-26

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PCT/EP2008/053580 WO2008116887A2 (fr) 2007-03-26 2008-03-26 Utilisation de mastocytes péritonéaux comme source d'héparine
PCT/EP2008/053582 WO2008116889A1 (fr) 2007-03-26 2008-03-26 Nouveau modèle in vitro de mastocytes de type séreux matures servant à l'analyse de maladies autoimmunes et d'inflammations allergiques

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PCT/EP2008/053582 WO2008116889A1 (fr) 2007-03-26 2008-03-26 Nouveau modèle in vitro de mastocytes de type séreux matures servant à l'analyse de maladies autoimmunes et d'inflammations allergiques

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CA3008848A1 (fr) 2015-12-18 2017-06-22 Tega Therapeutics, Inc. Compositions de glycosaminoglycane cellulaire et leurs procedes de preparation et d'utilisation
CA3152781A1 (fr) * 2019-08-27 2021-03-04 Tega Therapeutics, Inc. Heparine et sulfate d'heparane issus de cellules mst modifiees et procedes de fabrication et d'utilisation
CN114854688B (zh) * 2022-04-27 2023-09-26 广州医科大学附属第一医院（广州呼吸中心） 一种提高肥大细胞外泌体及其膜表面FcεRI的表达量的方法及其应用

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WO2004092356A2 (fr) * 2003-04-14 2004-10-28 Aventis Pharma S.A. Procede d'obtention de lignees de mastocytes a partir de tissus de porcs et procede de production de molecules de type heparine

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WO2004092356A2 (fr) * 2003-04-14 2004-10-28 Aventis Pharma S.A. Procede d'obtention de lignees de mastocytes a partir de tissus de porcs et procede de production de molecules de type heparine

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