fluticasone propionate-induced regulation of the balance within macrophage subpopulations
TRANSCRIPT
Fluticasone propionate-induced regulation of the balancewithin macrophage subpopulations
V. J. TORMEY, S. BERNARD, K. IVORY, C. M. BURKE* & L. W. POULTERDepartment of Immunology,Royal Free & University College School of Medicine, London, UK, and*Department of Respiratory Medicine,
James Connolly Memorial Hospital, Dublin, Ireland
(Accepted for publication 7 September 1999)
SUMMARY
In asthma, treatment with inhaled corticosteroids reduces chronic peribronchial inflammation andrestores the balance within macrophage subpopulations. This study investigates whether corticosteroidscan regulate monocyte differentiationin vitro and thereby influence the balance of functionally distinctmacrophages. Graded doses of fluticasone propionate (FP) were added to cultures of normal peripheralblood monocytes in the presence or absence of IL-4. Cells were harvested after 7 days’ culture. Doubleimmunofluorescence studies were performed on cytospins of differentiated macrophages using theMoAbs RFD1 and RFD7 to distinguish inductive and suppressive macrophages by their respectivephenotypes. Macrophage function was determined by quantifying allostimulation in a mixed leucocytereaction and by measuring tumour necrosis factor-alpha (TNF-a) production. FP reduced the number ofmature cells with a D1þ antigen-presenting phenotype and up-regulated the development of cells withthe D1/D7þ and D7þ phenotypes. Functionally, this was associated with reduced stimulation of T cellproliferation in a mixed leucocyte reaction (MLR). Fluticasone also reversed the increase in both D1þ
expression and TNF-a production induced by IL-4. The effect of FP persisted for 24 h after removal ofFP from the culture medium. These results suggest that FP treatment of asthmatics may have a directbeneficial effect by normalizing the macrophage subset imbalance that contributes to the chronicperibronchial inflammation present in this condition.
Keywords macrophage differentiation corticosteroids
INTRODUCTION
Within the heterogeneous population of pulmonary macrophagesthere are subsets of cells with the capacity to induce T cellresponses (dendritic antigen-presenting cells (APC)); subsetswith the capacity to suppress T cell responses (suppressive macro-phages); and phagocytic effector cells [1–3]. These subsets may bephenotypically discriminated using the MoAbs RFD1 and RFD7,and can be functionally discriminatedin vitro [3–5]. RFD1recognizes an epitope within the MHC class II complex whichappears restricted to APC [6] and RFD7 identifies a predominantlycytoplasmic antigen of 77 kD associated with mature phagocytes[6,7]. It has been shown within this laboratory and in manyindependent laboratories [8–11] that the use of these two reagentsin combination allows the discrimination of three subsets ofmacrophages with the respected phenotypes RFD1þ/RFD7¹,RFD1¹/RFD7þ, and RFD1þ/RFD7þ. In studies where these
populations have been investigated in relatively homogeneouspopulations, the D1þ phenotype has been consistently associatedwith the function of inducing T cell responsiveness, as demon-strated by enhanced T cell transformation in both allogeneic andantigen-driven responses. In contrast, the D7þ population showpredominantly a phagocytic function and the double-positive D1/D7þ have been shown to function as regulatory cells suppressing Tcell responsiveness [3,12].
Within the respiratory tract, T cells are tightly regulated byintrinsic and acquired immunosuppressive mechanisms whichnormally prevent T cell activation to non-pathogenic antigens[13]. Extensive studies have revealed that this regulation of Tcell stimulation in the lung may be controlled by suppressivemacrophages [2,14]. In asthma there is a state of immunedysregulation with chronic T cell-mediated peribronchialinflammation [15,16]. Analysis of the immunopathology ofendobronchial biopsies from asthmatic subjects reveals animbalance within these functionally distinct macrophage popula-tions, in that reduced proportions of suppressive cells areassociated with a chronic infiltrate of T cell macrophages andeosinophils [17].
Clin Exp Immunol 2000;119:4–10
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Correspondence: Dr V. J. Tormey, Department of Immunology, RoyalFree and University College School of Medicine, Rowland Hill St, LondonNW3 2PF, UK.
E-mail: [email protected]
In atopic asthmatics the T cell infiltrate is predominantly of theTh2 subset with cytokine mRNA for IL-4 and IL-5 identified inCD3þ cells obtained by bronchoalveolar lavage [18,19], andpresent in bronchial biopsies [20]. Complementary to its role insupporting IgE synthesis, IL-4 has been demonstrated to beessential to the commitment of naive CD4þ T cells to the Th2phenotypein vitro [21] and in vivo [22,23]. Furthermore, IL-4alters the balance within macrophage populations by increasing theproportion of D1þ inductive cells at the expense of D7þ and D1/D7þ effector and suppressive cells [4].
In asthma, therapeutic use of inhaled steroids reduces thenumber of infiltrating T cells, macrophages, dendritic cells, eosi-nophils and mast cells in the airway submucosa [24–26]. Not onlyis the total number of lung macrophages reduced but efficacioustherapy is associated with an alteration in the balance betweenphenotypically and functionally distinct macrophage subsets [25–27]. More specifically, in asthma there is a reduction in theproportion of D1þ inductive cells with an increase in the D7þ
effector cells and D1/D7þ suppressive cells [26].Previous studies have shown that T cell cytokines such as
interferon-gamma (IFN-g), IL-4, and IL-10 exert a significanteffect on mature macrophage phenotype and on differentiatingmonocytes [28–30]. It remains unclear therefore whether thechanges seen within the lung macrophage pool of steroid-treatedpatients are a direct effect on the monocyte/macrophage popula-tions or whether they are secondary to other immunomodulatingeffects of steroid therapy. The present study investigates in acontrolled in vitro environment the direct effect of fluticasonepropionate (FP) in regulating the balance within macrophagesubpopulations and its potential to modify the aberrant effect ofIL-4 on monocyte differentiation.
MATERIALS AND METHODS
Monocyte harvestPeripheral blood was obtained from normal healthy subjects byvenepuncture. Mononuclear cells were separated by density cen-trifugation (Nycomed Pharma AS, Oslo, Norway) at 650g for15 min. These were washed with PBS three times and suspended ata density of 1×106 cells/ml in RPMI 1640 culture medium (Sigma-Aldrich Co., Poole, UK) supplemented with 10% heat-inactivatedfetal bovine serum (FBS), 1·25% penicillin/streptomycin and1·25% 200 mM glutamine. Aliquots (2 ml) were then transferredto each well of 24-well culture plates. The cultures were incubatedat 378C in 5% CO2 to separate monocytes by adherence. After 2 hthe non-adherent cells were removed by aspiration and each wellwas washed three times in PBS preheated to 378C. Serum-free AIMV medium (2 ml) supplemented with 2×10¹5
M 2-mercaptoethanol(2-ME) was then added to each well. For each culture experimenttriplicate wells were harvested at this time (¼ T0). The method ofharvest is described below. The cell populations at T0 containedconsistently>90% monocytes as determined by morphology; theremainder of the cells were predominantly B cells. Viabilitydetermined by trypan blue exclusion was consistently>95%.The sensitivity of this separation technique was verified byimmunophenotyping with CD14 and CD68 as described in aprevious study [4].
Cell cultureAdherent monocytes were cultured in 24-well plates in AIM Vmedia (see above) for 7 days either with no addition or with the
addition of FP (Glaxo-Wellcome, Greenford, UK). In someexperiments dexamethasone (10¹5
M) was used to demonstratethat the observations made were a steroid class effect and notunique to fluticasone. Dose–response and time-course experimentswere carried out with addition of fluticasone at different concen-trations (10¹10–10¹5
M) and at different times (day 0 to day 6)during the 7-day culture period. All were added in 20-ml aliquotswith control cultures receiving 20ml of sterile PBS. All solutionsadded were warmed to 378C before addition. Cultures were allharvested after 7 days. At time of harvest plates were placed at 48Cfor 30 min and then vigorously aspirated with fresh cold PBS. Allcells from the wells were collected, including any cells no longeradhering to the plastic substrate. Cells were counted, viability wasreassessed and only cultures with a viability> 90% were used foranalysis. Experiments to determine dose–response were performedtwice.
Persistence of fluticasone effect on macrophage phenotypefollowing its removal from the culture media was determined bythe following method. Cell culture was performed as describedabove with the addition of fluticasone added on day 5. On day 7 cellswere washed twice in AIM 5, resuspended in the culture media andincubated at 378C. Cells were then harvested after 1, 3, 5, 7 and 24 h.
In some experiments recombinant human IL-4 (R&D Systems,Abingdon, UK) was added in 20-ml aliquots to the corticosteroid-treated cell cultures on day 5. Time-course and dose–responseeffect for cytokine addition has been reported previously. Controlcultures received 20ml sterile PBS.
Cytospin preparationAfter harvest at day 7 the cells were washed with PBS and centrifugedat 650g for 5 min. The cell density was adjusted to 3–5×105 cells/mland cytospins were prepared by spinning 50-ml aliquots at 80g for2 min in a Shandon cytocentrifuge (Shandon Southern Products Ltd,Runcorn, UK). Cytospins were air-dried for 1 h and fixed in a 1:1mixture of chloroform acetone for 10 min. These were then wrappedin cling film and stored at¹208C until analysed.
ImmunofluorescenceThe proportions of mature macrophage subsets within the har-vested cell populations were determined by double immunofluor-escence methods in which MoAbs RFD1 (mouse IgM) and RFD7(mouse IgG1 (Royal Free Hospital School of Medicine, London,UK)) were used in combination [31]. These reagents have beenextensively used in this laboratory and by many independentworkers to discriminate phenotypically distinct macrophage sub-sets. (They are commercially available from Serotec UK Ltd,Kidlington, UK). By using two immunoglobulin class-specificsecond layer reagents conjugated, respectively, to FITC andtetraethyl rhodamine isothiocyanate (TRITC; Europath Ltd,Bude, UK), the relative proportions of RFD1þ (hereafter referredto as D1þ) stimulating cells, RFD7þ (referred to as D7þ)phagocytes, and double-labelled RFD1þRFD7þ (referred to asD1/D7þ) suppressive cells could be determined.
These MoAbs were diluted 1:5 in PBS. Aliquots of 50ml wereapplied to the cytospin and incubated for 45 min in a moistchamber as above. Following incubation, the slides were washedtwice for 2 min in PBS. The second layer reagents were diluted1:50 in PBS and aliquots of 50ml were applied to the cytospinswhich were incubated for a further 45 min. The second layer wasremoved by washing twice (2×2 min) in PBS and the slidesmounted in PBS glycerol (9:1 dilution).
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Background staining or autofluorescence were identified bycomparison of test cytospins with control samples in which theprimary layer reagent was omitted. Non-specific staining byMoAbs was checked at standardization by comparison with thestaining produced by isotype-matched irrelevant MoAbs. Sectionsof human tonsil were used as positive controls.
The proportions of D1þ, D7þ and D1/D7þ fluorescent cellswere quantified by counting multiple high powered fields using aZeiss fluorescence microscope with epi-illumination and appro-priate barrier filters for FITC and TRITC.
For each subset the proportion of the cells was calculated by theformula:
ðspecific subsetÞðD1þÞ þ ðD7þÞ þ ðD1=D7þÞ
× 100
Cytokine ELISAThe production of tumour necrosis factor-alpha (TNF-a) andtransforming growth factor-beta (TGF-b) by differentiating mono-cytes was determined by cytokine-specific ELISA. The ELISA kitswere obtained from R&D Systems and had sensitivities down to15·6 pg/ml for both TNF-a and TGF-b.
Mixed leucocyte reactionsMeasurement of DNA synthetic rate by the incorporation of3H-thymidine has become a standard method for the measurement oflymphocyte proliferation in mixed leucocyte reactions (MLR)[32]. In some experiments the peripheral blood monocytes culturedfor 7 days in medium alone or with IFN-g or IL-10 added on day 5were harvested and treated with mitomycin C (Kyowa Ltd,London, UK). Mitomycin-C was used to stop cell division instimulator cell populations and to create one-way MLR whenstimulator and responder (with no mitomycin-C added) cell popu-lations were mixed [33].
Cells were incubated in the presence of mitomycin-C (50mg/ml) for 30 min at 378C in 5% humidified CO2. Cells were thenwashed three times in RPMI. Aliquots of 2×105 cells wereintroduced in triplicate into 96-well microtitre plates and co-cultured with autologous or allogeneic T cells added to each wellat a final concentration of 1×106 cells/well. The final volume ofculture medium was 200ml/well. This co-culture was incubated for6 days; this time period was selected on the basis of previous time-course experiments in this laboratory [34]. Eighteen hours beforetermination of cultures 1mCi 3H-thymidine (Amersham Ltd,Aylesbury, UK) was added to each well. Cells were harvestedusing an automatic cell harvester (Titer-Tek Flow, Laborat Inc.,McLean, VA) and counted in a liquid scintillation counter.
The average ct/min (of triplicate wells) expressed by theautologous cultures (without treatment of monocytes) werereduced to unity and all other cultures were expressed as astimulation index (SI) calculated by the factor whereby this wasgreater than or less than the untreated autologous MLR. This issummarized in the following equation: SI¼ [ct/min(monocytesþresponder peripheral blood mononuclear cells (PBMC)) – ct/min(responder PBMC)]/[ct/min(control monocytesþ autologousPBMC) – ct/min(responder PBMC)].
Statistical analysisThe effect of corticosteroids on monocyte maturation was analysedusing a paired non-parametric Mann–Whitney test.
RESULTS
Fluticasone alters the phenotype of differentiating monocytesAfter 7 days culture with no cytokine addition, 39% of cells wereD1þ, 40% of cells D7þ and 24% of cells D1/D7. The addition offluticasone on day 5 reduced the proportion of D1þ cells to 9%(P<0·05) and increased the proportion of D7þ up to 80%. Thiseffect was dose-dependent and could be produced even at a concen-tration of fluticasone of 10¹10
M (Fig. 1). When fluticasone was addedat progressive times from day 0 to day 6, its effect of increasingproportions of D7þ and D1/D7þ while reducing proportions of D1þ
cells did not change, except that when fluticasone was added on day 6(24 h before harvest) the effects were reduced (data not shown).
In vitro pharmacokinetics of fluticasoneTo demonstrate thein vitro pharmacokinetics of FP in relation toinhibitory effects on macrophage phenotype, a time-course experi-ment was performed in which macrophage phenotype was deter-mined at sequential time intervals following removal of FP fromthe culture. The inhibitory effect of FP persisted to 7 h post-contact. By 24 h however the effects began to wane (Fig. 2).
The effect of dexamethasone on monocyte differentiationThis inhibitory effect on macrophage differentiation is not uniqueto fluticasone but is shared by structurally different corticosteroids.The addition of dexamethasone at an optimum concentration of10¹5
M resulted in a reduction in the proportion of D1þ cells with aconcomitant increase in the proportion of cells expressing RFD7(D7þ plus D1/D7þ) (Fig. 3).
Fluticasone reverses the stimulatory effect of IL-4Following the present study of the inhibitory effect of corticosteroidson normal differentiating monocytes it remained to be determined
6 V. J. Tormeyet al.
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Fig. 1. Effect of fluticasone propionate (FP) concentration on macrophage phenotype. Dose–response effect of FP concentration on therelative proportions of monocytes expressing the stimulatory phenotype D1þ; phagocytic phenotype D7þ; and suppressive phenotype D1/D7þ after 7 days of culture. Fluticasone addition was made on day 5. Results represent mean6 s.e.m.
whether fluticasone could correct the dysregulation of monocytedevelopment previously shown to be induced by IL-4 [4]. IL-4increased the proportion of D1þ cells from 44% to 63% whilereducing the proportion of cells expressing the D7þ phenotype.The concomitant addition of fluticasone reversed this effect ofIL-4, with the proportion of D1þ cells decreasing from 63% to 36%and D7þ cells increasing from 35% to 62% (Fig. 4).
Effect of fluticasone on TNF-a and TGF-b production bydifferentiating monocytesSeven-day supernatants from all cultures were tested for levels ofcytokines TNF-a and TGF-b. Despite a trend towards decreasedTGF-b production induced by fluticasone and IL-4, this did notreach statistical significance (P> 0·05) (Fig. 5a). Fluticasone hadno direct effect on TNF-a production. However, IL-4 increasedTNF-a production and this was inhibited by the concomitantaddition of fluticasone (Fig. 5b).
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Fig. 2.Duration of fluticasone propionate (FP) effect on macrophage phenotype. Time-course experiment in which the relative proportions ofD1þ, D7þ and D1/D7þ macrophages were determined at sequential time intervals following removal of FP from the cell culture. FP additionwas made on day 5. Experiment performed in duplicate. Results of typical experiment shown.
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Fig. 3. Effect of dexamethasone on macrophage phenotype. Dexametha-sone addition was made on day 5. Experiment performed in duplicate.Results of typical experiment are shown.
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Fig. 4. Effect of fluticasone propionate (FP) and IL-4 in combination onmacrophage phenotype. The proportions of monocytes expressing thestimulatory phenotype D1þ; phagocytic phenotype D7þ; and suppressivephenotype D1/D7þ after 7 days culture in the absence or presence of FPand/or IL-4 added on day 5. Experiment performed in duplicate. Results oftypical experiment are shown.
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Fig. 5. (a) Effect of fluticasone propionate (FP) on transforming growthfactor-beta (TGF-b) production by differentiating monocytes. Resultsrepresent mean6 s.e.m. (b). Effect of FP on tumour necrosis factor-alpha(TNF-a) production by differentiating monocytes. Results represent mean6 s.e.m.
Modulation of monocyte differentiation by corticosteroids hasfunctional significanceMonocytes cultured in the presence of fluticasone for 48 h wereharvested at day 7 and admixed with allogeneic PBMC. Treatmentof the monocyte stimulator population with fluticasone signifi-cantly reduced T cell proliferation by 64% (Fig. 6). Thus thefluticasone-induced reduction in D1þ cells and increase in D7þ
cells significantly inhibited the T cell stimulatory capacity ofthe macrophage pool. Dexamethasone-treated monocytes alsoinhibited T cell proliferation in an allogeneic MLR (Fig. 6).
Monocytes cultured in the presence of fluticasone and IL-4were harvested at day 7 and admixed with allogeneic PBMC. Cellstreated with IL-4 promoted a non-significant increase in allogeneicMLR SI, which was inhibited by concomitant administration offluticasone (Fig. 6).
DISCUSSION
It has previously been shown [35] that corticosteroids alter thephenotype of mature alveolar macrophages of normal individuals.The present study extends these observations by demonstrating thatcontact with corticosteroids has a selective modifying effect onboth the phenotype and function of differentiating monocytes. Asthe subsets of cells distinguished by different phenotypes havebeen shown to exhibit different functions [3,34], the alteration incell phenotype caused by steroid contact here is important inunderstanding the mode of action of these therapeutic agents.However, since the monocyte population is 90% pure, a T celleffect, however small, may contribute as a confounding variable.
The capacity of fluticasone to down-regulate inductive macro-phages and increase effector and suppressive macrophages isconsistent with its known effectsin vivo. As well as altering thebalance between macrophage subpopulations itsin situ effects arecharacterized by the reduction in T cell-dominated chronic peri-bronchial inflammation. Similarly, in the presentin vitro study,changes in balance between macrophage subpopulations wereaccompanied by reduced antigen presentation (D1þ expression)
and hence lymphocyte proliferation. These inhibitory effects arenot unique to fluticasone but also shared by dexamethasone, albeitrequiring a higher concentration. Similar effects of glucocorticoidsin down-regulating dendritic cell functionin vivoandin vitro havebeen described previously [36].
These phenotypic and functional changes induced by flutica-sone are not associated with increased TGF-b production, althoughthis cytokine is known to be a potent inhibitor of T cell function[37,38]. This is consistent with other studies in which the inhibitionof T cell proliferation by IL-10-treated monocytes was notmediated by TGF-b [39].
It is well known that steroids can effect the release of cytokines[24,40–46] and may thus affect macrophage function. Corticoster-oids have recently been shown to down-regulate antigen-inducedgene expression for IL-4, IL-5, IL-13 and IFN-g [47] and thuspotentially have a profound indirect effect on macrophagephenotype and function. However, this study demonstrates thatfluticasone also inhibits the capacity of IL-4 to promote differ-entiation of D1þ inductive macrophages. The resulting shift inbalance within macrophage subpopulations towards D7þ effectorand D1/D7þ suppressive cells inhibits T cell function. Thus steroidsmay break the cycle of inflammation regulated by IL-4. This is ofparticular relevance to asthma because of the increased expression ofIL-4 mRNA within the bronchial wall [20]. Furthermore,in vitrostimulation of PBMC by allergen (Dermatophagoides pteronyssinus)increases IL-4 production [48].
The present study provides an important insight into thein vitropharmacokinetics of fluticasone. It is effective in determining themature macrophage phenotype when added at various time pointsduring monocyte differentiation. This is relevant to its therapeuticefficacy, because it is likely that there is a continuous turnover ofrecruited monocytes within the asthmatic bronchial wall. Thepersistence of a steroid effect for 24 h after removal of fluticasonefrom the cell culture is compatible with a high affinity for steroidreceptors within monocytes/macrophages, a feature which maycontribute to its potencyin vivo.
Thus the present results are indirect evidence that corticoster-oids may have a significant effect on those immunopathologicalsituations where chronic inflammation is associated with animbalance of macrophage subsets, such as asthma or inflammatorydisease [49]. They also provide indirect insight into the signifi-cance of the balance between inductive and suppressive macro-phages. Normally pulmonary macrophages down-regulate T cellimmune responses within the lower respiratory tract, thus main-taining local immunological homeostasis. It has been suggestedthat there is a cause and effect relationship between the reducedcapacity of local macrophage populations to suppress T cellactivity and the chronic nature of T cell-mediated inflammation[2,17]. Indeed, the alveolar macrophage-induced suppression of Tcell-induced hyperresponsiveness in asthma is reversed by allergenexposure [50]. However, corticosteroids reduce chronic peribron-chial inflammation while concomitantly correcting the dysregula-tion of monocyte differentiation. The capacity of an efficacious drugto inhibit lymphocyte proliferation by altering the balance withinmacrophage subpopulations is in itself indirect evidence for theimportance of the macrophage in regulating inflammation in asthma.
ACKNOWLEDGMENT
This work was supported by a grant from Glaxo Wellcome Research &Development.
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Control IL-4 FPFGST̀aHIUVIJVWDex
SI
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Fig. 6. Effect on steroid-treated monocytes on T cell proliferation. Theeffect of corticosteroids and IL-4 on T cell proliferation in an allogeneicmixed leucocyte reaction (MLR). The results are expressed as ct/min of3H-thymidine. Experiment performed three times, bars represent mean6 s.e.m. of stimulation index (SI). (Ct/min for allogeneic reactivity with-out cytokine addition were reduced to unity and all other results arerepresented as a SI in relation to this result.)
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