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WO1993019770A1 - Compositions permettant le traitement d'inflammations aigues ou chroniques - Google Patents

Compositions permettant le traitement d'inflammations aigues ou chroniques Download PDF

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WO1993019770A1
WO1993019770A1 PCT/GB1993/000719 GB9300719W WO9319770A1 WO 1993019770 A1 WO1993019770 A1 WO 1993019770A1 GB 9300719 W GB9300719 W GB 9300719W WO 9319770 A1 WO9319770 A1 WO 9319770A1
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macrophages
macrophage
tnf
expression
cells
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Siamon Gordon
Michael Stein
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Isis Innovation Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2026—IL-4

Definitions

  • the mononuclear phagocyte system has a central role in innate immunity, would healing and tissue homeostasis and, in addition, is intimately involved in the development and expression of specific immune responses.
  • the MPS is comprised of widely distributed tissue macrophages, circulating monocytes and, in inflamed tissues, recruited macrophages.
  • Bone-marrow derived monocytes, the immature precursors of the MPS, are highly adaptable cells and acquire specialised properties within local and distinct tissue environments.
  • the regulation of the macrophage ' s diverse functional repertoire, with its extraordinary secretory and endocytic potential, is of significant immunologic interest.
  • Tissue injury sets in motion a cascade of events designed to eliminate infectious agents and to maximise early reconstitution of the tissues.
  • tne earliest cells recruited to sites of tissue injury are neutrophils and macrophages.
  • Monocytes are rapidly directed from the circulation to the inflammatory site.
  • Macrophages elicited under such conditions are primed for a role in inflammation with altered expression of receptors and secretory products, but have not acquired maximal microbicidal activity.
  • Subsequent exposure of elicited macrophages to "activating" T lymphocyte-derived cytokines, expressed at the site of inflammation, is crucial to the development of fully microbicidal macrophages.
  • lymphokine activated cells from those cells with an elicited phenotype.
  • Foreign particles for example bacteria, that gain entry into the tissues are rapidly ingested by phagocytic cells, largely resident marcrophages.
  • Two major events may ensue; (1) the production of inflammatory mediators leading to recruitment of immune cells, including neutrophils and monocytes, and (2) the processing of ingested material for possible presentation to antigen-specific T-lymphocytes.
  • tissue cells or early elicited neutrophils and macrophages kill and degrade the invading microorganisms and the acute inflammatory stimulus is therefore removed.
  • lymphoid organs Activated T-helper cells at the inflammatory site and within stimulated lymphoid organs produce a large number of soluble immunoregulatory substances, many of which may have profound influence on macrophage antigen presenting function as well as effector function. Lymphokines act on newly recruited monocytes/macrophages to enhance their host-defense function, a crucial event for effective elimination of many pathogens.
  • macrophage activating factor/s a variety of assays, in particular assays of respiratory burst activity, were used to measure the effect of cellular supernatants on human or mouse macrophage populations (Simon and Sheagren, 1972).
  • IFN-g accounted for many features of the activated macrophage phenotype by "conventional" criteria including enhanced MHC class 2 antigen (Ag)expression, priming for subsequent triggering of the respiratory burst, and enhanced microbicidal activity. It was also demonstrated that IFN-g was capable of inducing macrophage tumoricidal activity (Schreiber et al, 1986).
  • cytokines have direct or synergistic macrophage activating properties.
  • GM-CSF granulocyte- macrophage colony stimulating factor
  • monocytes a particularly potent activator of monocytes, (Reed et al, 1987).
  • cytokines can be regarded as potent and direct macrophage immunologic activators although some may be important "accessory" molecules.
  • the process of immunologic activation is complex, involving the expression and regulation of multiple gene products.
  • the use of killing mechanisms is only partly useful in defining the activated macrophage phenotype, and more simple antigen-based assays that reflect this process are required for in situ studies of immunologic activation.
  • Table 1 below shows ways in which iramunologically activated cells are distinguished from resting cells (circulating monocytes or tissue macrophages) or elicited cells (attracted to an inflammatory focus by non-specific stimuli), by altered expression of specific markers.
  • MHC class 2 Ag expression MHC class 2 Ag expression
  • MMR macrophage mannose receptor
  • Other as yet uncharacterised surface antigens have been proposed as markers of macrophage activation (Mackay et al, 1989).
  • la (MHC class II) expression gives an overall assessment of exposure to immunogen and lymphokine.
  • Assays of MMR differentiate resident or elicited macrophages from activated macrophages while assays of H 2 0_ generation discriminate between immunologically unstimulated (resident and elicited) and activated populations.
  • IGF-1 Insulin-like growth factor 1
  • GH growth hormone
  • the MMR is a lectin-like endocytic receptor which binds mannosylated glycoproteins and particles. It has a large number of possible physiologic and microbial ligands and phagocytic potential that together suggest a likely role in host defense.
  • the MMR is a useful marker of macrophage maturation (Ezekowitz and Stahl, 1988). It is not expressed on freshly isolated human monocytes, but following in vitro culture its expression is induced co-incident with the acquisition of macrophage endocytic and secretory functions, for example increased phagocytic activity of Fc and complement receptors, and enhanced secretion of lysosomal enzymes. MMR expression reaches a stable peak after 3-5 days in culture.
  • TGF-b1 and -b2 T-cell growth factor b1 and b2
  • MDF macrophage deactivating factor
  • MDF has been purified to near homogeneity but its relationship to other molecules, for example IL-10 (interleukin 10), is unknown, although a recent report suggests it is distinct (Bogdan et al, 1991).
  • MDAFs in particular TGF-b, and PGE_ (Prostaglandin E_) (Davies et al, 1980)
  • macrophages themselves may have a built in negative feedback mechanism for decreasing pro- inflammatory activity. MDAFs have convincingly been shown to induce or potently enhance the release of growth promoting factors or protease inhibitors.
  • An established model of macrophage immunologic activation involves the notion of macrophage activation/de-activation as a linear but reversible pathway from the circulating monocyte, through inflammatory recruitment, towards maximal microbicidal and tumoricidal activity. This traditional view has become too narrow to accommodate the functional diversity of cytokine actions, but remains useful in highlighting the adaptive capacity of macrophages to respond to micro-evnironmental signals. Further, the use of specific sets of in vitro assays as indicators of immunologic activation is helpful in defining a role for a particular set of cytokines within various models of immunity. The ability of macrophages to penetrate areas of inflammation and to respond rapidly to local conditions is crucial to their role in host defense.
  • cytokines now described is long and growing and macrophages, at least in tissue culture, are capable of producing a large number of these.
  • the local regulation of macrophage secretion is likely to be very important in determining the physiological role of macrophages and knowledge in this area is essential for understanding and manipulation of host defenses.
  • T- helper subsets expressing mutually inhibitory cytokine profiles have been demonstrated in various mammalian models of infection.
  • Th-1 and Th-2 cells two functionally distinct T-helper (Th) cell populations, Th-1 and Th-2 cells, have been well characterized.
  • Th-1 cells secrete IL-2, IFN-g and lymphotoxin/TNF (tumour necrosis factor) .
  • Th-2 cells do not express IL-2, IFN-g or TNF but secrete high levels of IL-4, IL-5, IL-6 and IL-10. Both subsets produce I -3 and GM-CSF (reviewed by Mosmann and Coffman, 1991). Functionally distinct helper T-cell subsets correlate well with the development of different immune responses. For example, in mice, Th-1 cells are associated with cell-mediated immunity and DTH (delayed-type hypersensitivity) reactions, while Th-2 cells promote humoral immunity. In addition, effective host-defense against some infections appears to be dependent on the balance between Th-1 and Th-2- derived lymphokine production.
  • Th-1 cell clones derived from resistant C57/BL6 mice adoptively transferred to susceptible Balb/c mice provide protection against lethal infection with Leishmania major.
  • Th-2 derived cytokines exacerbate infection in C57/BL6 animals.
  • Th-2 cell responses In humans a similar association of Th-2 cell responses with failure of effective cell-mediated killing of a pathogen has been made in patients with lepromatous leprosy (Yamamura et al, 1991). Th subsets have not been elucidated in humans to the same extent as in rodents but recent results show human T- cell clones with the lymphokine profiles of murine Th cells (Salgame et al, 1991).
  • the balance of cytokine production by different T-helper cells and macrophages is likely to be crucial to elimination of the pathogen and return of the inflammatory focus to the normal state. Delayed-type hypersensitivity reactions on the one hand are impressive examples of immune imbalances leading to excessive macrophage inflammatory activity and tissue damage, while on the other hand, the anergy of lepromatous leprosy of miliary tuberculosis patients are examples of inadequate host-defense. Although the effects of IFN-g and TNF on macrophages have been well characterised, the influence of Th-2 cytokines on macrophage function is poorly understood.
  • IL-4 has apparently contradictory effects on macrophage host defense function, profoundly suppressing many activities, for example inflammatory cytokine release, while stimulating variable expression of MHC class 2 antigen on different macrophage populations.
  • the definitive roles of IL-4 in regulating macrophage immune function are therefore unclear, as are those of I -10.
  • IL-10 is a potent inhibitor of macrophage dependent antigen presentation to Th-1 cells.
  • it inhibits inflammatory cytokine production by an IL-4, TGF-b and MDF independent mechanism, inhibits IFN-g priming of the respiratory burst and suppresses Th-1 subset cytokine secretion. Therefore, it is likely to powerfully suppress delayed type hypersensitivity reactions.
  • Th-2 cells stimulates MHC class 2 ag expression on B-cells and promotes cytotoxic T-cell capability.
  • the present invention is based on the discovery that stimulation of elicited macrophages (BgPM) with Th-2 supernatant or TH-2 derived lymphokines, IL-4 and IL-10, with or without IL-5 and IL-6, results in a clearly distinguishable phenotype, herein called a healing phenotype, with dramatically altered endocytic and secretory properties in comparison with IFN-g activated macrophages.
  • a healing phenotype herein called a healing phenotype
  • a further aspect of the invention is a composition for use in the treatment of an acute or chronic inflammation which comprises supernatant of macrophages with a healing phenotype.
  • the macrophages may have a healing phenotype as a result of treatment with a mixture comprising Th-2 cell supernatant, and/or as a result of treatment with a mixture comprising IL-4 and 11-10.
  • the invention further provides a method of treatment of an acute or chronic inflammation comprising applying supernatant of macrophages with a healing phenotype to the inflammation to promote or enhance tissue repair.
  • Another method according to the invention is the treatment of an acute or chronic inflammation comprising applying IL-4 and IL-10 to the inflammation to promote or enhance tissue repair.
  • the invention also provides a method of treatment of an acute or chronic inflammation comprising applying IL-4 and IL-10 to the inflammation to promote or enhance tissue repair.
  • Another aspect of the invention is a method of producing macrophages with a healing phenotype in vitro by contacting a population of macrophages with a composition comprising IL-4 and IL-10, and/or Th-2 cell supernatant.
  • a further aspect is a method of promoting healing which comprises applying a mixture of IL-4 and IL-10 to an acute or chronic inflammation to produce macrophages with a healing phenotype.
  • Also provided by the invention is a method of identifying the physiological mediators of wound repair by identifying the products of macrophages with a healing phenotype and testing the products for their healing effect.
  • Th-1 (KLH ag specific) and Th-2 (conalbumin ag specific) clones were obtained from Dr. T. Mosmann (DNAX Research Institute, Palo Alto, Ca.). The Th-2 clone was lost after the second passage and the Th-1 clones failed to produce IFN-g following 3 weeks in culture. Therefore, stored cell supernatants and subsequently combinations of recombinant proteins that correspond to the appropriate T-h cell subset were used for these studies.
  • mice were bred and housed at the Sir William Dunn School of Pathology, University of Oxford. In general, adult male Balb/c mice were used between age 8-12 weeks for experiments.
  • Dulbecco ' s modified Minimum essential medium (DMEM) , RPMI and OPTIMEM, a serum-free proprietary reagent, were obtained from Gibco-Biocult Ltd., Paisley, Scotland.
  • Fetal bovine serum (FBS) was obtained from Seralab UK, Crawley Down, Wales, England and routinely heat inactivated for 30 minutes at 56°C. Media were supplemented with 10% foetal bovine serum (FBS) , glutamine (2mM) , penicillin (50 ⁇ g/ml) and streptomycin (100 ⁇ g/ml).
  • Phosphate- buffered saline (PBS) pH-7.4 was obtained from Oxoid Ltd. Basingstoke, U.K.
  • LPS E.Coli 0111.B4
  • zymosan A and mannan both from Saccharomyces cerevisiae
  • Sigma Chemical company St. Louis, Mo.
  • the following highly purified recombinant murine proteins were gifts from the indicated source.
  • Interferon gamma (5 x 10 U/ml) from Dr. F. Balkwill, ICRF, London, U.K. IL-4 from Dr. S. Gillis, Immunex Corporation, Seattle, Ca, U.S.A. TNF alpha from Dr. E. Havell, Trudeau Institute, Saranac Lake, U.S.A.
  • GM-CSF from Dr. M. Mielke, Freie Universiteit, Berlin, Germany.
  • M-CSF and IL-3 from British Biotechnology, Oxford, U.K. IL-6 from Dr. Van Snick, Leuven University, Belgium.
  • IL-10 from Dr. Kevin Moore, DNAX Research Institute, Palo Alto, Ca., U.S.A. Human TGF beta from Dr. J. Heath, Oxford University, U.K. Human RANTES and MlP-la from Dr. T. Schall, Genentech Inc., San Francisco, Ca., U.S.A.
  • Macrophages were isolated from the mouse peritoneal cavity. Thioglycollate broth (1ml), proteose-peptone solution (1ml of a 1% solution) and Bio-Gel (biogel) bead-elicited macrophages were isolated 4 or 5 days after intra-peritoneal injection. Biogel beads were washed by repeated centrifugation, autoclaved and -1 ml of a 2% v/v suspension injected ip. Cells were routinely plated at 3 x 10 macrophages/well in 24 well tissue culture plates. The cells were incubated for 1 hour at 37°C in a 5% C02 incubator and then washed 4x with PBS at 4°C to remove non-adherent cells.
  • the cells were treated within 12 hours of harvest unless otherwise indicated, when adherent monolayers consisted of >90% macrophages, and viability was >97% by phase contrast microscopy and trypan blue exclusion.
  • adherent monolayers consisted of >90% macrophages, and viability was >97% by phase contrast microscopy and trypan blue exclusion.
  • BgPM Bio-Gel bead- elicited peritoneal macrophages
  • 10cm bacterial plastic plates as before but left in RPMI, routinely supplemented with 10 mM glutamine, 10% FBS, penicillin and streptomycin, overnight.
  • the cells became non-adherent and were easily washed off the dishes. These cells were centrifuged, resuspended
  • Each 1 ml fraction was carefully layered into a 10 ml conical centrifuge tube to make stepped gradients of the following densities (g/ml) : (1) 1.100, (2) 1.090, (3) 1.080, (4) 1.075, (5) 1.070, (6) 1.060.
  • the macrophage fraction was present at the interface of fractions 5 and 6 (ficoll method) or at the metrizamide/medium interface (metrizamide method) .
  • the macrophage fraction >99.5% pure by immunocytochemistry (F4/80 and FA.11 ag analysis), phase microscopy and latex ingestion) was collected and re-plated before cytokine treatment. RNA was isolated as described later.
  • TNF levels in macrophage culture supernatants were measured using a modified mouse L929 fibroblast toxicity assay as previously described by Flick and Gifford, 1984. L929 cells were routinely tested and shown to be free of mycoplasma.
  • Actinomycin D (1 ⁇ g/ml) treated cells were incubated with test samples for 18 hours. The cells were washed, fixed in methanol and stained with crystal violet (1% w/v)/methanol. Changes in absorbance at 510 nm were quantitated by a Dynatech MR600 microtiter plate reader (Dynatech Laboratories, Alexandria, VA, USA) and converted to units per millilitre based on a standard curve using murine rTNF. To compare TNF release by different M ⁇ populations, 3 x 10 M ⁇ were plated in each well and cell protein determined as required. A blocking anti-TNF antibody was routinely used with each sample to ensure that all L929 cell death was due to TNF.
  • I- mannose-BSA is detectable after »40 minutes incubation at 37°C and continues at a linear rate for several days if M ⁇ are maintained in the continuous presence of ligand. Trace amounts of sterile ligand (»10 C.P.M. in 10 ⁇ l) were added to monolayers of adherent M ⁇ populations. Cells were incubated for 16 hours (unless otherwise indicated) and a 0.4 ml aliquot of medium removed to microfuge tubes.
  • Trichloroacetic acid was added to a final concentration of 10% w/v, the tubes incubated on ice for 30 minutes and then spun for 10 minutes in a centrifuge. Supernatant (0.2 ml) was removed and 5 ⁇ l of potassium iodide (4M) followed by 10 ⁇ l of H_0 2 were added to each aliquot, incubated for 10 minutes at room temperature, followed by the addition of 0.8 ml of chloroform. The mixture was vortexed vigorously, spun and 100 ⁇ l of the clear aqueous phase assayed in a gamma counter. Cell- dependent, saturable degradation of 125I-mannose-BSA per unit time was calculated as a function of M ⁇ number. Cell-free blanks were used routinely.
  • Superoxide anion release assay measures the change in colour of cytochrome C when reduced by superoxide anion released from the stimulated macrophage.
  • the assay described below is based on the assay described by Johnston, R.B. et al, 1978.
  • Macrophages were plated at 1-3 x 10 /well in RPMI 1640, 10% FCS or OPTIMEM (as indicated in figure legends) in a 24 well tissue culture plate. The density ensures that oxygen anion release is proportional to cell number. Monolayers were washed in Hanks balanced salt solution (HBSS; Gibco) without phenol red. The cells were pre-incubated for 5 minutes in a reaction mixture containing: - Hanks buffered saline solution (HBSS)
  • Negative controls contained bovine superoxide dismutase (Sigma Chemical Co., type I) final concentration of 30 ⁇ g/ml. After 5 minutes, stimuli were added for 60 minutes. Stimuli: Phorbol myristate acetate (PMA) (Sigma Chemical Co.) was added to a , final concentration of 20 - 100 ng/ml. PMA was maintained at -70 C in a stock solution of 2mg/ml in dimethylsulpoxide (DMSO) (Siga Chemical Co.). DMSO alone did not stimulate cytochrome C reduction by cultured cells. In some cases zymosan (32-64 ⁇ g/ml) was used as an additional stimulus.
  • PMA Phorbol myristate acetate
  • DMSO dimethylsulpoxide
  • Lysozyme activity was measured using the "lysoplate” assay described by Osserman and Lawlor (1966). Micrococcus lysodeikticus cell walls (Sigma Chemical Co.) were suspended at 0.3mg/ml in 1% molten agarose in 10mM Tris-HC1 pH 7.0, and poured into 10 cm diameter petri dishes. Wells were punched into the set agar plates, and lysozyme containing samples placed in the wells (20 ⁇ l per well) . A clear zone of lysis around the well, appearing after 4-8 hours, indicated lysozyme activity. For quantisation, the radius of the zone of lysis was measured, and a standard curve constructed showing radius of lysis/hr versus ⁇ g of hen egg lysozyme (Sigma Chemical Co.), in the range 0.03 ⁇ g to 10 ⁇ g.
  • BgPM were harvested, plated (3 x 10 macrophages per well) and washed prior to co-incubation with Th-1 or Th-2 lymphocyte clone conditioned medium or combinations of IFN-g (50U/ml) and TNF (500U/ml), or
  • IL-4 (1ng/ml) and IL-10 (10OU/ml) as indicated.
  • Dexamethasone (DEX) was used at 10 M. Monolayers were incubated for 48 hours prior to addition of 125I- g mannose-BSA (6 10 cpm/ ⁇ g) with or without excess unlabelled mannose-BSA. Following overnight incubation, supernatants were removed for degradation analysis as in Materials and Methods. Where sequential addition of lymphokines was necessary (IL>IFN-g: IFN-g->IL-4) , monolayers were incubated in the presence of the primary stimulus, washed, and the secondary stimulus added for the following 24 hours prior to the addition of ligand. Results are displayed in figure 1 and show mean +/- SD of pooled results of three independent experiments.
  • Th-lymphokine stimulated BgPM gene expression RT-PCR analysis. (see figure 2)
  • TNF, MMR, IGF-1 and lysozyme mRNA transcripts by BgPM purified by differential density centrifugation 1.5 x 10 macrophages (99.8% pure by F4/80 ag immunocytochemistry) were plated in OPTIMEM in 24 well plates, and incubated in the presence or absence of either IFN-g (50U/ml) and TNF (500U/ml), or IL-4 (1ng/ml) and IL-10 (10OU/ml) for 48 - 56 hours.
  • Control monolayers were mock-treated with PBS. Separate sets of monolayers (right hand side of the figure) were further stimulated with LPS (lug/ml) for 2 hours.
  • A TNF, MMR and lysozyme (LYZ) .
  • B IGF-1 (IGF) and lysozyme.
  • a and B represent independent experiments.
  • Input cDNA was representative of about 50 cell equivalents per lane except for lysozyme PCR products shown in B (bottom panel) , where input cDNA was representative of 20 cell equivalents.
  • Photographic exposure times were not constant, as may be assessed by the difference in intensity of the various marker lanes.
  • IGF-1 gene expression conducted as described in the legend to figure 2 except indicated monolayers were incubated in the presence of LPS for 1.5 hours.
  • op panel IL- 1
  • Bottom panel MMR and IGF- 1 expression, in the absence and presence of LPS , respectively, were assayed in parallel and are shown for reference .
  • Adherent BgPM express MCP-1 transcripts.
  • Adherent BgPM (3 x 10 per well) were incubated for 56 hours in the presence of: IL-4 (1ng/ml)(+) IL-4 + IL-10, IFN-g, or medium (OPTIMEM) alone(o) . Except for IL-4 (alone) (+) and medium control (o) , indicated as single data points, lymphokines were used at 10-fold dilutions with starting concentrations as follows: IL-4 (1ng/ml) and IL-10 (50U/ml); IFN-g (10OU/ml). LPS (1 ⁇ g/ml), PMA
  • TNF release was measured by L929 bioassay, following 16 hours LPS or zymosan stimulation.
  • Middle panel 0_ release assayed 1 hour following PMA (100ng/ml) or zymosan (64 ⁇ g/ml stimulation.
  • Superoxide dismutase 25 ⁇ g/ml inhibited all 0- reduction of ferricytochrome C. Results are expressed as nmol 0_/mg cell protein/60 minutes.
  • mice The effect of Th-2 macrophage conditioned medium on inflammatory sites was tested in mice. Nude mice were shaved on the back and a 2cm long scratch incision was made on each. Two groups were treated at the site of the incision with the following:
  • the liquids were applied in a gauze swob and left for 24 hours, after which time the wounds were observed. The swobs were replaced and the wounds were observed again at 72 hours.
  • IL-5 and IL-6 alone or in combination with IL-4, had no apparent effect on MMR expression (not shown) .
  • Th-1 lymphokine only IFN-g had a directly inhibitory effect on MMR expression, although TNF blocked IL-4 dependent enhancement.
  • Figure 1 demonstrates the cross-regulatory influence of Th-1 and Th-2 derived cytokines in which IFN-g or TNF completely inhibit the effect of IL-4 on MMR activity. In the presence of saturating doses of both IL-4 and IFN-g, no enhancement of MMR activity occurs. However, if IFN-g is added more than 24 hours after IL-4 it does not fully reverse the enhanced MMR expression within the following 48 hour period.
  • MMR activity of secondarily IFN-g treated BgPM was 84% of the MMR levels of control cells (IL-4 treated, no secondary stimulus) .
  • IL-4 treated, no secondary stimulus IL-4 treated, no secondary stimulus
  • M-CSF moderately (2-fold) enhanced MMR expression.
  • IL-3 produced by both Th-1 and Th-2 cells, induced cell division, but did not increase MMR expression when expression was corrected for cell number (not shown) .
  • Lysozyme gene expression is unaffected by either treatment. There is a minimum 16-fold difference between the IL-4/IL-10 and the IFN- g/TNF groups with respect to IGF-1 and MMR expression as measured by titrations of input cDNA (not shown) . MCP-1 gene expression was similarly inhibited by IL- 4/IL-10 and enhanced by IFN-g/TNF (fig. 3 - bottom panel). LPS induced TNF and IL-1 mRNA to an apparently similar extent in control cells, IL-4/IL-10 and IFN-g/TNF treated groups. Bioassay for TNF release following LPS (1 ⁇ g/ml) stimulation confirmed the TNF mRNA data, at the protein level (fig. 4 - top panel) .
  • results of respiratory burst analysis of the two populations are compared with the effect on TNF release and la expression (fig. 4).
  • IL-4/IL-10 suppresses PMA triggered 0_ release.
  • IL-4 alone has no effect on PMA triggered respiratory burst (not shown), but apparently enhances zymosan triggered 0- release.
  • IFN-g primes BgPM for subsequent phagocytic or PMA induced 0_ release, as previously shown (Nathan et al, 1983).
  • IFN-g in combination with IL-10 failed to prime cells for subsequent PMA trigger of release (not shown).
  • IL-4/IL-10 was inhibitory for PMA triggered 0- release, but had less effect on zymosan induced release.
  • TNF release and la expression are inhibited by IL-4/IL-10 treatment and enhanced by IFN-g stimulation. TNF secretion following zymosan challenge was only partially suppressed by IL-10/IL-4 stimulation.
  • Indirect-binding assays demonstrate the inhibition of la expression by IL-4/IL-10 in comparison to its enhancement by IFN-g.
  • IFN-g/TNF treatment in the absc ce of a further trigger, enhances expression of IL-1 and TNF expression.
  • LPS challenge following LPS challenge,
  • IL-4/IL-10 treatment did not measurably inhibit TNF or IL-1 mRNA levels of TNF or IL-1.
  • IL-4/IL-10 treatment clearly blocks release of bioactive TNF; and northern blots (results not shown) indicate that both murine and human IL-4, and human IL-10, inhibit expression of TNF following LPS challenge.
  • PCR is probably too sensitive to detect the relatively smaller differences in expression of IL-1 and TNF as compared to IGF-1 and MMR.
  • TNF mRNA levels are post transcriptionally regulated (Beutler et al, 1986) and it would not have been surprising to find an apparent discrepancy between mRNA levels and protein secreted.
  • IL-4/IL-10 inhibits la expression on elicited mouse macrophages.
  • Th- 1 activated BgPM low MMR and IGF-1; high la and TNF
  • Th-2 activated macrophages high MMR and IGF-1, low la and TNF
  • Th-2 derived cytokines provide an alternative pathway for macrophage activation as compared to the immunologic activation that occurs during infections in which IFN-g activates macrophages.
  • FIG. 5 An extended model of macrophage immunologic activation is presented in fig. 5, in which 1 represents Enhanced microbicidal activity against facultative i/c pathogens; 2 represents Immunologic activation; 3 represents possible specific microbicidal function, and repair functions.
  • the model illustrates the possible influence of soluble and/or surface interactions of Th-1 and Th-2 lymphocytes on macrophage function within inflammatory infiltrates. Th-1 and Th-2 lymphokine may act sequentially or simultaneously on macrophage function, therefore macrophage deactivation is not necessarily a step-wise phenomenon as implied in the previous model.
  • IL-10 may be a powerful inhibitor of la expression and PMA triggered 0- generation, and by extension an inhibitor of killing of some intracellular pathogens, Th-2 activated macrophages may have as yet undiscovered killing function for specific pathogens.
  • Th-1 and Th-2 macrophage phenotypes in vivo correlates and functions
  • Th-1 macrophages are phenotypically equivalent to BCG- or IFN-g activated macrophages and probably have a role in cell-mediated immune responses and DTH reactions.
  • Th-2 macrophages recruited to the inflammatory site may acquire maximal and specialised host-defense function against specific pathogens, for example mannosylated microorganisms, or parasites which evoke an IgE humoral response.
  • the Th- 2 macrophage may be used as a potent scavenger cell for the clearance of damaged cells bearing exposed mannose residues, or other as yet uncharacterised carbohydrate ligands for the MMR or CD 23.
  • Th-2 macrophages may further promote wound healing by inhibiting the release of inflammatory stimuli and enhancing release of IGF-1, and probably other growth factors, which act on stromal cells within the local environment to promote their growth and secretion of matrix components.
  • Elicited macrophages may therefore be differentially stimulated to alter their function according to signals derived from different Th cell subsets.
  • Th-1 and Th-2 macrophage phenotypes characterised here are easily distinguished by differential expression of endocytic and secretory markers and may represent extremes or only two parts of the elcitied macrophage immunologic modulation spectrum in vivo (fig. 5 - dashed lines indicate hypothetical pathways of feedback regulation of Th cellfunction) .
  • This heterogeneity in macrophage immunologic activation emphasizes the unique host defense potential of the MPS.

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Abstract

On décrit une composition qui permet le traitement d'inflammations aiguës ou chroniques et comprend IL-4 et IL-10, ou un surnageant de cellules Th-2, ou un surnageant de macrophages dotés d'un phénotype favorisant la guérison. On décrit en outre un traitement correspondant. Les macrophages dotés d'un phénotype favorisant la guérison constituent un groupe de macrophages récemment définis et dérivés de macrophages produits stimulés par IL-4 et IL-10 ou par un surnageant de cellules Th-2, et ils présentent un phénotype nettement différencié.
PCT/GB1993/000719 1992-04-06 1993-04-06 Compositions permettant le traitement d'inflammations aigues ou chroniques WO1993019770A1 (fr)

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GB929207732A GB9207732D0 (en) 1992-04-06 1992-04-06 Wound repair

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US6706264B1 (en) 1994-03-14 2004-03-16 Genetics Institute, Llc Use of IL-12 antagonists in the treatment of conditions promoted by an increase in levels of IFN-y
US6737550B2 (en) 2001-06-13 2004-05-18 Biogal Gryogyszergyar Rt. Process for preparing rac-bicalutamide and its intermediates
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US7102026B2 (en) 2001-06-13 2006-09-05 Teva Gyógyszergyár Zártkörüen Müködö Részvénytársaság Process for preparing and isolating rac-bicalutamide and its intermediates
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WO2014004465A1 (fr) * 2012-06-25 2014-01-03 The Brigham And Women's Hospital, Inc. Thérapie ciblée
US9790264B2 (en) 2012-06-25 2017-10-17 The Brigham And Women's Hospital, Inc. Compounds and methods for modulating pharmacokinetics
US10851143B2 (en) 2011-11-08 2020-12-01 Synerkine Pharma B.V. Methods of treatment with a fusion protein comprising IL-4 and IL-10
US11312757B2 (en) 2019-04-19 2022-04-26 Synerkine Pharma B.V. Fusion protein comprising IL13

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US6123915A (en) * 1990-08-02 2000-09-26 Fred Hutchinson Cancer Research Center Methods for using agents that bind to VCAM-1
US7449186B1 (en) 1990-08-02 2008-11-11 Fred Hutchinson Cancer Research Center Methods of blocking the interaction between stromal cells and hemopoietic cells with anti-VCAM-1 antibodies
US5827670A (en) * 1990-08-02 1998-10-27 Fred Hutchinson Cancer Research Center Methods of isolating and detecting bone marrow stromal cells with VCAM-1-specific antibodies
US6706264B1 (en) 1994-03-14 2004-03-16 Genetics Institute, Llc Use of IL-12 antagonists in the treatment of conditions promoted by an increase in levels of IFN-y
US6338848B1 (en) * 1994-03-14 2002-01-15 Genetics Institute, Inc. Use of IL-12 and IL-12 antagonists in the treatment of autoimmune diseases
US7138115B2 (en) 1994-03-14 2006-11-21 Genetics Institute, Llc Use of IL-12 and IL-12 antagonists in the treatment of autoimmune diseases
US8012475B2 (en) 1994-03-14 2011-09-06 Genetics Institute, Llc Use of IL-12 and IL-12 antagonists in the treatment of autoimmune diseases
US7534430B2 (en) 1994-03-14 2009-05-19 Genetics Institute, Llc Use of IL-12 and IL-12 antagonists in the treatment of autoimmune diseases
US6830751B1 (en) 1994-03-14 2004-12-14 Genetics Institute, Llc Use of IL-12 antagonists in the treatment of rheumatoid arthritis
US6387364B1 (en) 1995-08-09 2002-05-14 Renovo Limited Methods of healing wounds and fibrotic disorders using IL-10
GB2304047A (en) * 1995-08-09 1997-03-12 Univ Manchester Pharmaceutical compositions containing cytokines
US7052684B2 (en) 1995-08-09 2006-05-30 Renovo Limited Methods of healing wounds and fibrotic disorders using IL-10
WO1997005894A1 (fr) * 1995-08-09 1997-02-20 The Victoria University Of Manchester Composition pharmaceutique contenant il-10
US6797263B2 (en) 2000-05-12 2004-09-28 Beth Israel Deaconess Medical Center, Inc. Compositions and methods for achieving immune suppression
US7347995B2 (en) 2000-05-12 2008-03-25 Beth Israel Deaconess Medical Center, Inc. Compositions and methods for achieving immune suppression
US7102026B2 (en) 2001-06-13 2006-09-05 Teva Gyógyszergyár Zártkörüen Müködö Részvénytársaság Process for preparing and isolating rac-bicalutamide and its intermediates
US6849763B2 (en) 2001-06-13 2005-02-01 BIOGAL Gyógyszergyár Rt. Process for preparing rac-bicalutamide and its intermediates
US6797843B2 (en) 2001-06-13 2004-09-28 BIOGAL Gyógyszergyár Rt. Process for preparing rac-bicalutamide and its intermediates
US6737550B2 (en) 2001-06-13 2004-05-18 Biogal Gryogyszergyar Rt. Process for preparing rac-bicalutamide and its intermediates
US6861557B2 (en) 2001-06-13 2005-03-01 BIOGAL Gyógyszergyár Rt. Process for preparing rac-bicalutamide and its intermediates
EP2452688A1 (fr) * 2010-11-12 2012-05-16 Ruprecht-Karls-Universität Heidelberg Utilisation de macrophages transfectés par l'ARN 10 interleukine dans des thérapies anti-inflammatoires
WO2012062930A1 (fr) * 2010-11-12 2012-05-18 Ruprecht-Karls-Univeristät Heidelberg Utilisation de macrophages transfectés par arnm d'interleukine 10 dans des thérapies anti-inflammatoires
US9315558B2 (en) 2010-11-12 2016-04-19 Ruprecht-Karls-Universität Heidelberg Use of interleukin 10 mRNA transfected macrophages in anti-inflammatory therapies
US10851143B2 (en) 2011-11-08 2020-12-01 Synerkine Pharma B.V. Methods of treatment with a fusion protein comprising IL-4 and IL-10
US10981964B2 (en) 2011-11-08 2021-04-20 Synerkine Pharma B.V. Fusion protein comprising IL-4 and IL-10
WO2014004465A1 (fr) * 2012-06-25 2014-01-03 The Brigham And Women's Hospital, Inc. Thérapie ciblée
US9790264B2 (en) 2012-06-25 2017-10-17 The Brigham And Women's Hospital, Inc. Compounds and methods for modulating pharmacokinetics
US9421245B2 (en) 2012-06-25 2016-08-23 The Brigham And Women's Hospital, Inc. Targeted therapeutics
US11312757B2 (en) 2019-04-19 2022-04-26 Synerkine Pharma B.V. Fusion protein comprising IL13

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