WO1993009797A1

WO1993009797A1 - Treatment of macrophages

Info

Publication number: WO1993009797A1
Application number: PCT/GB1992/002110
Authority: WO
Inventors: Siamon Gordon; Michael Stein; Satish Keshav
Original assignee: Isis Innovation Limited
Priority date: 1991-11-15
Filing date: 1992-11-13
Publication date: 1993-05-27
Also published as: GB9124347D0; AU2913892A

Abstract

This invention is based on the discovery that Interleukin-4 (IL-4) greatly enhances macrophage mannose receptor activity. The invention provides use of IL-4 or an IL-4 antagonist or IL-4 receptor blocking agent in the treatment of human or animal patients suffering from infections involving mannosylated pathogens.

Description

TREATMENT OF MACROPHAGES

IN RQPUCTION

The macrophage mannose receptor (MMR)

(previously called the mannosyl fucosyl receptor (MFR) is an important phagocytic receptor mediating the binding and ingestion of micro-organisms with surface mannose residues and soluble mannose-containing glycoproteins. It is expressed on resident and elicited peritoneal and alveolar macrophages but not on monocytes, and at very low level on BCG-activated

1 macrophages ( ) . Therefore, the MMR is a marker of the resident and elicited but immunologically non-activated macrophage phenotype. MMR activity is decreased by gamma interferon, increased by steroids and beta- interferon, and inversely correlated with major histocompatibility (MHC) class 2 antigen expression which is induced on immunologically stimulated macrophages and used as a marker of activation.

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

2 as a macrophage activating factor ( ) . However, the 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. In contrast, gamma interferon induces all three class 2

3 molecules ( ) . Furthermore, 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 ( ) . In addition, 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) . Lastly, 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.

IPVEETIQN

In one aspect, 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. This method may be performed on macrophage cells in vitro, that is to say outside the living body, or alternatively in vivo.

In another aspect 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. For example, 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. Alternatively, 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

10 the discussion section below. 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. Formulations for

15 such purposes may be standard. For example, a cream or ointment may be used in the treatment of a vaginal or skin infection.

MATERIALS AND METHODS

20

Animals

Adult male Balb/C mice were bred at the Sir William Dunn School of Pathology, University of Oxford.

25

Media and reagents

RPMI was obtained from 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. Media

30 were supplemented with 10% FBS, glutamine (2 mM) , penicillin (50 μg/ml) and streptomycin (100 μg/ml). 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

35 Chemical Co. (St. Louis, Mo.). Antibodies and cytokines

11B11, 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

11 purified ( ) . Rec. murine interferon gamma was a gift from Dr. F. Balkwill, ICRF, London, U.K., and rec. murine L-4 was a gift from Dr. S. Gillis, Immunex, U.S.A.

Cells

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. 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.

Macrophage mannosyl receptor (MMR) assays

These assays were performed as described previously ( 12) with modifications as indicated.

Briefly, mannosylated-BSA (E-Y Laboratories, 127N

Amphlett Blvd., San Mateo, CA. ) was trace labelled with Na(125)I by a modified chloramine-T method. Ligand was tested for trichloroacetic acid (TCA) precipitability before use. f_*

Degrada ion Of I-mannose-BSA by macrophages (M0) was measured by the appearance of TCA-soluble labelled

12₅ material in the culture medium. Degradation of I- mannose-BSA is detectable after ^"40 min incubation at

37^*C and continues at a linear rate for several days if

M0 are maintained in the continuous presence of

10 ligand. Trace amounts of sterile ligand (^"10 c.p.m. in 10 μl) were added to monolayers of adherent Mβ populations. Cells were incubated for 16 hrs (unless otherwise indicated) and a 0.4 ml aliquot of medium removed to microfuge tubes. Trichloroacetic acid was

15 added to a final concentration of 10% w/v, the tubes incubated on ice for 30 min and then spun for 10 min in a centrifuge. Supernatant (0.2 ml) was removed and 5 μl of potassium iodide (4M) followed by 10 μl of H₂0₉ were added to each aliquot, incubated for 10 min at RT,

20 followed by the addition of 0.8 ml of chloroform. The mixture was vortexed vigorously, spun and 100 μl of the clear aqueous phase was counted in a gamma counter. Cell-dependent, saturable degradation of 125I-mannose- BSA per unit time was calculated as a function of M0

25 number. Cell-free blanks were routinely used.

Binding of mannose-specific liσands

Binding was assayed at saturating concentrations of ligand using trace labelled mannose- 30 "5

BSA (saturation 250 ng/ml ligand/5 x 10 M0) in the presence or absence of mannan (5 mg/ml) or 100 fold excess unlabelled mannose-BSA. Binding was assayed after one hour at 4'C. Cells were washed in ice-cold

PBS with 10 mM-sodium azide. Then 500 μl of 1 N NaOH 35 was added to dissolve the cells and the cell-associated radioactivity measured in a Packard gamma spectrometer (Packard Instrument Co. Inc., Downes Grove, IL, USA). Results were expressed as nanograms of mannose-BSA specifically bound or taken up per 5 x 10 M0 plated.

Semi-quantitative polymerase chain reaction (PCR) analysis

1 x 10⁶ BgPM, IL-4, gIFN treated or mock- treated as described in the figure legends, were washed once with PBS (4^*C) and lysed with 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). 1:100 dilution of cDNAs from each condition were subject to the polymerase chain reaction (PCR) (annealing temperature: 60^*C, (Mg ] = 2.0 mM) using the following oligonucleotide primers: MMR (unpublished sequence) Sense: AAA CAC AGA CTG ACC CTT

CCC; Antisense: GTT AGT GTA CCG CAC CCT CC) . Tumour necrosis factor (TΝF) ( 13) (Sense: TGG CAG AAG AGG CAC

TCC CC; Antisense: GAG GAG CAC GTA GTC GGG GC) .

Lysozyme (LYZ) (¹⁴) (Sense: CTA TGG AGT CAG CCT GCC G;

Antisense: CAT GCT CGA ATG CCT TGG GG) . PCR products were subjected to agarose gel electroporesis and visualised by ethidium bromide staining. The specificity of each amplification was verified by restriction enzyme cleavage of the product at an internal site.

Reference is directed to the accompanying drawings in which.-

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, gIFΝ and dexamethasone (Dex) . Cells 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 μg/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

Methods. Non-specific counts, as determined by the addition of 100 fold excess of mannose-BSA, were always

<15% of the total. The data shown are from 3 separate experiments done in duplicate.

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. RNA from control, gIFN and IL-4 treated

BgPM was reverse-transcribed, and cDNA fragments specific for MMR (top), lysozyme (middle) and TNF alpha

(bottom) were amplified, as described in Materials and

Methods. Lane 1 and 2 = IL-4 treated (16 hr and 4 hr respectively); lane 3 = gIFN treated (16 hr) ; lane 4

= control. Table 1

Time course of the effect of IL-4 on MMR activity;

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

(^"0.4 μg/ml) was added for the last 4 hours of the time course whereafter the amount of ligand degraded was measured as described in Materials and Methods.^'

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.

** For this time point the ligand was added for the entire incubation. 11B11 is an IL-4 blocking mAb. Table 2

Effect of IL-4 on zymosan uptake

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.

RESULTS:

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. Further, following three days in the presence of IL-4 occasional giant cells were noted. This effect depended on the plating density. Parallel cultures of the more widely used thioglycollate-elicited macrophage populations were not useful in assessing these changes since these cells are normally tightly adherent to tissue culture plastic under the culture conditions used here. These findings prompted further analysis of IL-4 modulation of the elicited macrophage phenotype.

Expression of the MMR is well established as a marker for the non-activated elicited or resident macrophage phenotype. As shown in Figure 1, 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. The changes in surface MMR expression were assessed by high-affinity binding at 4'C in the presence of labelled ligand in response to maximal doses of IL-4 (5 ng/ml) . Data in Figure 2 show that maximal and saturable binding was increased about 8 fold compared to control cultures. The apparent affinity of the receptor is approximately equal for both populations. Mannan or excess cold mannose-BSA effectively competed for binding and degradation of iodinated ligand. MMR and mRNA is present at only low levels and Northern blot analysis of mRNA induction was not successful. Therefore, reverse transcriptase PCR analysis of MMR mRNA levels was performed. 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 (lower panel) 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) .

Lastly, the 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.

DISCUSSION

IL-4 has been regarded as an activator of certain macrophage functions, such as tumoricidal

2 capacity and MHC class 2 expression ( ) . However, IL-4 also decreases the expression of specific pro- inflammatory cytokines, in apparent opposition to its role as an activator.

It has been demonstrated that 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.

The effect of IL-4 in up-regulating MMR plasma membrane activity is not a general phenomenon since IL-4 reduces CD14 expression (

. Taken together with the induced CR-3-dependent spreading and enhanced uptake of complement-opsonized sheep erythrocytes ( ), and the enhanced uptake of zymosan, a mannosylated yeast wall particle, it is likely that IL-4 may couple endosomal machinery to specific phagocytic plasma membrane receptors to promote their internalization.

Other cytokines tested so far include TGFb, IFΝb,^'TΝF, 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.

Recently, monomeric IgG2a was reported to induce MMR expression ( 17) . However, the IgG2a effect was studied in relation to bone marrow derived macrophage precursor maturation but not elicited monocyte/macrophage populations. Further, while 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. In addition, IL-4 enhances the release of IgG-1 and IgE, but greatly inhibits the release of IgG2a, from activated B-cells (¹⁸).

These data suggest that 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) .

Additional circumstantial evidence of an IL-4-like action on alveolar macrophages is their very low pro- inflammatory secretory activity.

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.

Indeed, 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. On the other hand, the role of 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.

REFERENCES!

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2. Crawford, R.M. , D.S. Finbloom, J. Ohara, w.E. Paul, and M.S. Meltzer. 1987. B cell stimulatory factor-1 (interleukin 4) activates macrophages for increased tumoricidal activity and expression of Ia antigens. J. Immunol . 139:135.

3. Gerrard, T.L., D.R. Dyer and H.S. Mostowski. 1990. 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.

4. Donnelly, R.P., M.J. Fenton, D.S. Finbloom and T.L. Gerrard. 1990. Differential regulation of IL-1 production in human monocytes by IFN-gamma and IL-4. J. Immunol . 145:569.

5. McBride, W.H., J.S. Economou, R. Nayersina, S. Comora and R. Essner. 1990. Influences of interleukins 2 and 4 on tumor necrosis factor production by murine mononuclear phagocytes. Cancer Res . 50: 2949-52

6. Standiford, T.J., R.M. Strieter, S. . Chensue, J. Westwick, K. Kasahara and S.L. Kunkel. 1990. IL-4 inhibits the expression of IL-8 from stimulated human monocytes. J. Immunol . 145: 1435

7. Hart, P.H., G.A. Whitty, D.R. Burgess, M. Croatto and J.A. Hamilton. 1990. Augmentation of glucocorticoid action on human monocytes by interleukin-4. Lymphokine Res . 9: 147

8. S.L. Abra son and J.I. Gallin. 1990. IL-4 inhibits superoxide production by human mononuclear phagocytes. J. Immunol. 144: 625

9. Phillips, .A., M. Croatto and J.A. Hamilton. 1990. Priming the macrophage respiratory burst with IL-4: enhancement with TNF-alpha but inhibition by IFN-gamma. Immunology. 70: 498

10. H.P. Tan, S.L. Nehlsen-Cannarella, CA. Garberoglio and J.M. Tosk. 1991. Recombinant interleukin-4 enhances the che-niluminescent oxidative burst of murine peritoneal macrophages. J.Leukoc.Biol . 49: 587 11. Rosen, H. and S. Gordon. 1987. Monoclonal antibody to the murine type 3 complement receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo. J. Exp. Med. 166: 1585

12. Stahl, P., P. Schlesinger, E. Sigardson, J.S. Rodman and Y.C. Lee. 1980. Receptor mediated pinocytosis of mannose glycoconjugates by macrophages, characterisation and evidence of recycling. Cell. 19: 207.

13. Pennica, D., J.S.Hayflick, T.S.Bringman, M.A.Palladino and D.Goeddel. 1985. Cloning and expression in Escherichia coli of the cDNA or murine tumor necrosis factor. Proc . Natn . Acad . Sci . U.S.A. 82: 6060.

14. Cross, M., I. Mangelsdorf, A.Wedel and R. Renkawitz. 1988. Mouse lysozyme Mgene: Isolation, characterization andexpression studies. Proc. Natl . Acad. Sci . U.S.A. 85: 6232.

15. Sampson, L.L., J. Heuser and E.J. Brown. 1991. Cytokine regulation of complement receptor-mediated ingestion by mouse peritoneal macrophages. M-CSF and IL-4 activate phagocytosis by a common mechanism requiring autostimulation by IFN-beta. J. Xizunimol. 146: 1005.

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Claims

1. A method of treating macrophages to alter their mannose receptor activity which method comprises contacting the macrophages with either interleukin-4 (IL-4) or an IL-4 antagonist or IL-4 receptor blocking 0 agent.

2. A method as claimed in claim 1, wherein the step of contacting the macrophages is performed in vitro.

3. Use of interleukin-4 (IL-4) or an IL-4 5 antagonist or IL-4 receptor blocking agent for the preparation of a medicament for treating infections involving mannosylated pathogens.

4. A method for the treatment of a human or animal patient suffering form an infection involving a 0 mannosylated pathogen, which method comprises administering to the patient either interleukin-4 (IL- 4) or an IL-4 antagonist or IL-4 receptor blocking agent.

5. A method as claimed in claim 4, wherein ^J administration is systemic.

6. A method as claimed in claim 4, wherein administration is topical.

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