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WO1993009797A1 - Traitement des macrophages - Google Patents

Traitement des macrophages Download PDF

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Publication number
WO1993009797A1
WO1993009797A1 PCT/GB1992/002110 GB9202110W WO9309797A1 WO 1993009797 A1 WO1993009797 A1 WO 1993009797A1 GB 9202110 W GB9202110 W GB 9202110W WO 9309797 A1 WO9309797 A1 WO 9309797A1
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Prior art keywords
macrophages
mmr
cells
macrophage
mannose
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PCT/GB1992/002110
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English (en)
Inventor
Siamon Gordon
Michael Stein
Satish Keshav
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Isis Innovation Limited
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Publication date
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Publication of WO1993009797A1 publication Critical patent/WO1993009797A1/fr

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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]
    • A61K38/2026—IL-4

Definitions

  • MMR macrophage mannose receptor
  • MFR mannosyl fucosyl receptor
  • MMR macrophages
  • Interleukin-4 (IL-4), predominantly produced by activated T-helper cells of the type 2 phenotype has pleiotropic effects on a variety of immune and non-immune cells. As it induces the expression of MHC class 2 antigen on B-cells and monocytes and enhances macrophage tumoricidal activity it has been described
  • tumoricidal activity is restricted to selected target cell lines and only HLA-DR and HLA-DP but not HLA-DQ
  • MHC class 2 molecules are induced by IL-4.
  • gamma interferon induces all three class 2
  • IL-4 inhibits the expression of pro-inflammatory cytokine genes such as interleukin 1 (IL-1), tumour necrosis factor and IL-8 and synergizes with steroids to inhibit macrophage
  • a C C_ *7 pro-inflammatory activity ( ) .
  • IL-4 inhibits superoxide anion release from pyrimidine myristyl acetate or zymosan treated monocytes ( ) although this effect depends critically on the particular macrophage source used and the presence of other cytokines ( 9' 10) .
  • IL-4 treated human monocytes express acid phosphatase, a marker of macrophage maturation in vitro. 2-3 days sooner than o untreated cells ( ) .
  • This invention is based on the discovery that IL-4 greatly enhances MMR activity of murine peritoneal exudate macrophages.
  • the potency and efficacy of IL-4 is unmatched by any other known MMR inducer, such as the better known macrophage deactivating agent, dexamethasone.
  • the data taken together with previous studies indicate that IL-4. induces elicited macrophages to adopt an alternative macrophage phenotype, with very high MMR activity, restricted MHC class 2 antigen expression and reduced pro-inflammatory cytokine secretion.
  • the invention provides a method of treating macrophages to alter their mannose receptor activity which method comprises contacting the macrophages with either interleukin 4 (IL-4) or and IL-4 antagonist or IL-4 receptor blocking agent.
  • IL-4 interleukin 4
  • IL-4 antagonist or IL-4 receptor blocking agent.
  • This method may be performed on macrophage cells in vitro, that is to say outside the living body, or alternatively in vivo.
  • the invention concerns the treatment of a human or animal patient suffering from an infection involving mannosylated pathogen.
  • Mannosylated pathogens are known to include a variety of yeasts and fungi including Candida species and Saccharomvces cerevisiae.
  • yeasts and fungi including Candida species and Saccharomvces cerevisiae.
  • Pneumocystis carinii is a mannosylated pathogen now known to be a yeast. ft.
  • Treatment may involve the use of interleukin-4 (IL-4) normally in order to increase macrophage mannose receptor activity.
  • IL-4 interleukin-4
  • treatment may involve the use of an IL-4 antagonist or IL-4 receptor blocking agent, normally in order to reduce macrophage mannose receptor activity. The significance of these two alternatives is addressed in
  • IL-4, IL-4 antagonists and IL-4 receptor blocking agents are all known and available materials (e.g. from Immunex, U.S.A.). Administration of the agent may be by any known technique, e.g. systemic or topical.
  • a cream or ointment may be used in the treatment of a vaginal or skin infection.
  • mice Male male Balb/C mice were bred at the Sir William Dunn School of Pathology, University of Oxford.
  • RPMI Gibco-Biocult Ltd., Paisley, Scotland.
  • Fetal bovine serum (FBS) was obtained from S-eralab UK Ltd., CrawleyDown, U.K. and routinely heat inactivated for 30 min at 56'C.
  • Biogel P100 fine was obtained from Bio-rad Laboratories, Richmond, CA, U.S.A. Mannan and zy osan (from Saccharomyces cerevisiae) was obtained from Sigma
  • an IL-4 blocking rat monoclonal antibody was purified from an ATCC hybridoma cell line obtained through Dr. W. E. Paul (NIH, Bethesda, Maryland, U.S.A.). 5C6, a mouse complement receptor (CR3) blocking rat monoclonal antibody was isolated and
  • Rec. murine interferon gamma was a gift from Dr. F. Balkwill, ICRF, London, U.K.
  • rec. murine L-4 was a gift from Dr. S. Gillis, Immunex, U.S.A.
  • Macrophages were isolated from the mouse peritoneal cavity. Thioglycollate-elicited and biogel bead-elicited macrophages were isolated 4-5 days after intra-peritoneal injection. Cells were plated at 3x10 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. Thereafter, cells were treated as described in the figure legends. For RNA isolation, Biogel-bead elicited peritoneal macrophages (BgPM) were incubated in 10 cm bacterial plastic plates as before but left in RPMI with 10% FBS overnight.
  • BgPM Biogel-bead elicited peritoneal macrophages
  • the cells become non-adherent and are easily washed off the dishes. Following centrifugation the cells were spun into a Percoll (Pharmacia) differential density gradient. The macrophage fraction (>99.5%) pure by immunocytochemistry) was collected and re-plated before cytokine treatment.
  • MMR Macrophage mannosyl receptor
  • Binding was assayed at saturating concentrations of ligand using trace labelled mannose- 30 "5
  • RNAzol solution (Cinna/Biotecx laboratories, Texas, USA) .
  • Total RNA was isolated and reverse transcribed by standard procedures using Moloney Murine Leukaemia Virus reverse transcriptase (British Research Laboratories/GIBCO, UK).
  • T ⁇ F Tumour necrosis factor
  • Lysozyme ( 14 ) (Sense: CTA TGG AGT CAG CCT GCC G;
  • Antisense CAT GCT CGA ATG CCT TGG GG
  • Figure 1 is an MMR activity dose response curve showing degradation of 125I-mannose-BSA by BgPM in response to increasing doses of recombinant murine
  • IL-4, gIF ⁇ and dexamethasone (Dex) were harvested and plated in equal numbers per well as described in Materials and Methods. Cells were incubated in the continuous presence of IL-4, gIFN or Dex for 48 hours before the addition of 125-I-mannose- BSA (0.4 ⁇ g/ml). Specific TCA soluble counts present in the culture medium after 16 hours in the continuous presence of 125-I-mannose-BSA were used as a measure of ligand degradation as detailed in Materials and
  • Figure 2 is a ligand binding curve of BgPM incubated with increasing amounts of 125I-mannose-BSA.
  • BgPM were incubated with or without IL-4 (5 ng/ml) and 48 hours later specific binding of 125I-mannose-BSA was measured as described in Materials and Methods. Kd for control and IL-4-treated cells are similar, indicating that differences in 125I-mannose-BSA binding reveal changes in receptor capacity rather than affinity. The data shown represent one of two similar experiments done in triplicate.
  • Figure 3 shows expression of MMR, lysozyme and TNF mRNA transcripts by IL-4 treated peritoneal macrophages.
  • BgPM was reverse-transcribed, and cDNA fragments specific for MMR (top), lysozyme (middle) and TNF alpha
  • Monolayers of macrophages were cultured in medium, containing 10% FCS, for the time indicated in the presence or absence of IL-4.
  • 125-I-mannose-BSA 125-I-mannose-BSA
  • Results reflect the mean +/- SE of at least two separate experiments and are expressed as ngs of ligand degraded per 0.5 x 10 cells.
  • 11B11 is an IL-4 blocking mAb.
  • Monolayers of macrophages were cultured in medium, containing 10% FCS, for the time indicated in the presence or absence of gIFN or IL-4. Zymosan was added for the last 5 minutes of the time course. The cells were vigorously washed in ice-cold PBS and the monolayers incubated at 37'C for 15 min. Thereafter, the monolayers were washed again and the number of cell-associated particles were counted following lysis of the cells in water containing 0.1% triton X-100. Results are expressed as mean number of particles per cell and represent one of two similar experiments done in duplicate.
  • the elicited macrophage population used in this study facilitated analysis of the morphological effect of the various cytokine treatments. These cells become rounded and relatively non-adherent to tissue culture plastic following overnight incubation, a hitherto unpublished observation. Striking morphologic changes occurred within 8 hours following addition of IL-4. After overnight culture the cells became firmly adherent and spread out on the tissue culture plastic. This effect was more than 90% inhibitable by co- incubation of macrophages with 5C6, a rat anti-mouse CR-3 blocking mAb. Indirect binding assays for CR-3 using 5C6 showed only a small increase in surface CR-3 expression on IL-4 treated macrophages.
  • MMR MMR-activated elicited or resident macrophage phenotype.
  • maximal MMR activity after 48 hours culture in recombinant murine IL-4 was about 15 fold higher than in untreated controls.
  • Half-maximal induction occurred at an IL-4 concentration of less than 100 pg/ml.
  • Gamma interferon decreased and dexamethasone increased MMR activity.
  • Table 1 shows that maximal degradation activity occurs after 48 hours although increased activity was measurable after 8 hours of IL-4 (5 ng/ml) treatment. Addition of an anti-murine IL-4 mAb, 11B11, completely prevented the enhanced MMR activity.
  • Figure 3 shows that IL-4 increases MMR mRNA levels as assayed by the PCR using murine MMR specific oligonucleotides.
  • the unpublished sequence of a mouse MMR cDNA was kindly provided by Dr. Alan Ezekowitz, Harvard Medical School, Boston, USA.
  • the same IL-4 or gIFN treated BgPM cDNAs were analysed for lysozyme and TNF mRNA levels.
  • Figure 3 shows the small decrease in specific signal for TNF mRNA. Lysozyme mRNA levels were relatively unaltered by the various treatments.
  • the PCR data showing IL-4 dependent increase in MMR mRNA levels were confirmed by nuclease protection assays (data not shown) .
  • IL-4 effect on phagocytic function was assessed by zymosan uptake studies.
  • Table 2 shows that IL-4 treated macrophages bind and ingest about 4 fold more zymosan than control cells and about 8 fold more than gIFN treated cells.
  • IL-4 has been regarded as an activator of certain macrophage functions, such as tumoricidal
  • IL-4 also decreases the expression of specific pro- inflammatory cytokines, in apparent opposition to its role as an activator.
  • IL-4 potently enhances the expression and activity of the MMR ( Figures 1, 2), an important endocytic receptor known to mediate the binding and ingestion of mannosylated proteins and macromolecules. This effect was similarly demonstrated on thioglycollate-elicited macrophages, another elicited but immunologically non-activated macrophage population (data not shown) . The increased binding and activity is associated with increased MMR mRNA levels ( Figure 3) . Further, CR3 is "activated" by
  • IL-4 ( ) and that in addition, spreading on tissue culture plastic of previously non-adherent macrophages is largely CR3 dependent.
  • the modulation of the MMR, long used as marker for the immunologically non- activated macrophage phenotype, together with the down-regulation of numerous pro-inflammatory cytokines suggest that low and possibly physiologic concentrations of IL-4 are able to induce recently recruited monocytes to adopt an alternate phenotype not previously considered for inflammatory macrophages.
  • Such a cell may have maximal endocytic clearance capacity for mannosylated ligands but would be relatively quiescent with respect to pro-inflammatory cytokine production.
  • IL-4 in up-regulating MMR plasma membrane activity is not a general phenomenon since IL-4 reduces CD14 expression ( .
  • IL-4 reduces CD14 expression ( .
  • zymosan complement-opsonized sheep erythrocytes ( )
  • zymosan a mannosylated yeast wall particle
  • cytokines tested so far include TGFb, IF ⁇ b, ' T ⁇ F, IL-2, IL-6, GM-CSF, M-CSF and IL-10, but these recombinant proteins have only modest or no effect on elicited murine MMR activity in comparison to IL-4.
  • IgG2a monomeric IgG2a was reported to induce MMR expression ( 17) .
  • the IgG2a effect was studied in relation to bone marrow derived macrophage precursor maturation but not elicited monocyte/macrophage populations.
  • IgG2a greatly enhanced the early expression of MMR on BMM in culture, the maximum level of MMR activity was not greater than mock-treated cells incubated under standard conditions for 7 days. The effects leading to this invention were probably not due to IgG2a production by contaminating B-cells, as highly purified macrophage populations were used.
  • IL-4 enhances the release of IgG-1 and IgE, but greatly inhibits the release of IgG2a, from activated B-cells ( 18 ).
  • IL-4 is a candidate regulator of MMR expression in specific tissue micro- environments. Although maximal in vitro IL-4 stimulation induces at least 10 fold higher MMR activity than is present in resident peritoneal macrophages, low doses of IL-4 such as those found within tissues, may maintain MMR expression at high levels, for example on alveolar macrophages ( 19) .
  • Unbalanced production of IL-4 may enhance MMR activity and therefore cause excess uptake of mannosylated micro-organisms while inhibiting production of pro-inflammatory cytokines. This may retard the initiation of inflammatory cell recruitment, and may be particularly relevant to pathological states where there is already reduced pro-inflammatory cytokine production such as may occur in immunodeficiency diseases, for example HIV infection.
  • MMR has been reported to mediate the ingestion of pneumocvstis carinii by alveolar macrophages ( 20) and phagocytosis of unopsonised Candida species ( 21 ) .
  • IL-4 antagonists or IL-4 receptor blocking agents may be useful in the treatment or prevention of infections where excess pathogen is taken up through the macrophage mannose receptor.
  • IL-4 as a potent MMR inducing agent suggests it may be of use in enhancing expression of MMR in order to maximise clearance of mannosylated microorganisms, for example yeasts, as reflected by IL-4 treated macrophages having increased capacity to bind and ingest zymosan.
  • B cell stimulatory factor-1 (interleukin 4) activates macrophages for increased tumoricidal activity and expression of Ia antigens. J. Immunol . 139:135.
  • IL-4 and granulocyte-macrophage colony-stimulating factor selectively increase HLA-DR and HLA-DP antigens but not HLA-DQ antigens on human monocytes. J. I-n-nunol. 144:4670.
  • IL-4 inhibits the expression of IL-8 from stimulated human monocytes. J. Immunol . 145: 1435

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Abstract

L'invention est fondée sur la découverte que l'Interleukine-4 (IL-4) accroît considérablement l'activité du récepteur de mannose des macrophages. L'invention concerne l'utilisation de l'IL-4, ou d'un antagoniste d'IL-4, ou d'un agent bloquant le récepteur d'IL-4 dans le traitement d'infections comportant des agents pathogènes mannosylés chez les êtres humains ou les animaux.
PCT/GB1992/002110 1991-11-15 1992-11-13 Traitement des macrophages WO1993009797A1 (fr)

Applications Claiming Priority (2)

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GB9124347.7 1991-11-15
GB919124347A GB9124347D0 (en) 1991-11-15 1991-11-15 Treatment of macrophages

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004009A3 (fr) * 2000-07-12 2003-08-21 Immunex Corp Methode de traitement du cancer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991014450A1 (fr) * 1990-03-21 1991-10-03 Schering Corporation Utilisation de l'il-4 pour stimuler la reponse immunitaire aux attaques d'antigenes infectieux

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991014450A1 (fr) * 1990-03-21 1991-10-03 Schering Corporation Utilisation de l'il-4 pour stimuler la reponse immunitaire aux attaques d'antigenes infectieux

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY vol. 2, no. 4, April 1990, pages 317 - 318 P. D. STAHL 'The macrophage mannose receptor : Current status' cited in the application *
EUROPEAN JOURNAL OF IMMUNOLOGY vol. 20, 1990, pages 2375 - 2381 R. P. LAUENER ET AL 'Interleukin 4 down regulates the expression of CD14 in normal human monocyres' cited in the application *
PATHOBIOLOGY vol. 59, no. 4, August 1991, BASEL,CH. page 302 G. ROSSI ET AL 'Biphasic mode of mannose receptor expression' *
THE JOURNAL OF EXPERIMENTAL MEDICINE vol. 176, no. 1, 1 July 1992, NEW YORK pages 287 - 292 M. STEIN ET AL 'Interleukin 4 potently enhances murine macrophage mannose receptor activity : A marker of alternative immunologic macrophage activation' *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004009A3 (fr) * 2000-07-12 2003-08-21 Immunex Corp Methode de traitement du cancer

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AU2913892A (en) 1993-06-15

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