CAPITAL FUNDACIONAL

SUMMARY

In asthma treatment with inhaled corticosteroids reduces chronic peribronchial inflammation and restores the balance within macrophage subpopulations. The study in this chapter investigates whether corticosteroids can regulate monocyte differentiation in vitro and thereby influence the balance of functionally distinct macrophages in the tissues. Graded doses of fluticasone propionate (FP) were added cultures of normal peripheral blood monocytes in the presence or absence of lL-4. Cells were harvested after 7 days culture. Double immunofluorescence studies were performed on cytospins of differentiated macrophages using the monoclonal antibodies RFDl and RFD7 to distinguish inductive and suppressive macrophages by their respective phenotypes. Macrophage function was determined by quantifying allostimulation in a mixed leucocyte reaction and by measuring TNFa.

FP reduced the number of mature cells with a D1+ antigen-presenting phenotype and up-regulated the development of cells with the D1/D7+ and D7+ phenotypes. Functionally, this was associated with reduced stimulation of T cell proliferation in an MLR. Fluticasone also reversed the increase in both D1+ expression and TNFa production induced by lL-4. The effect of FP was thus similar to that of IL- 10 described previously. The effect of FP persisted for 24 hours after removal of FP from the culture medium. These results suggest that FP has a direct effect on the macrophage component of chronic peribronchial inflammation.

INTRODUCTION

Within the heterogenous population of pulmonary macrophages there are subsets of cells with the capacity to induce T cell responses such as dendritic antigen presenting cells, subsets with the capacity to suppress T cell responses (suppressive macrophages), and phagocytic effector cells (Toews et al 1984; Thepen et al 1994; Spiteri & Poulter 1991). These subsets may be phenotypically discriminated using the monoclonal antibodies RFDl and RFD7 and can be functionally discriminated in vitro (Spiteri & Poulter 1991; Tormey et al 1997; Poulter 1991; Poulter & Burke 1996; Hutter & Poulter 1992).

Within the respiratory tract, T cells are tightly regulated by intrinsic and acquired immunosuppressive mechanisms which normally prevent T cell activation to non-pathogenic antigens (Holt et al 1991). Extensive studies have revealed that this regulation of T cell stimulation in the lung may be controlled by suppressive macrophages (Thepen et al 1994; Strickland et al 1994). In asthma there is a state of immune dysregulation with chronic T cell mediated peribronchial inflammation (Poulter et al 1990; Beasley et al 1989). Analysis of the immunopathology of endobronchial biopsies from asthmatic subjects reveals an imbalance within these functionally distinct macrophage populations in that reduced proportions of suppressive cells are associated with a chronic infiltrate of T cells macrophages and eosinophils (Poulter et al 1994).

In atopic asthmatics the T cell infiltrate is predominantly of the Th2 subset with cytokine mRNA for IL-4 and IL-5 identified in CD3+ cells obtained by

bronchoalveolar lavage (Robinson et al 1992, et al 1993) and also on bronchial biopsies (Ying et al 1995). Complementary to its role in supporting IgE synthesis, IL-4 has been demonstrated to be essential to the commitment of naive CD4+ T cells to the Th2 phenotype in vitro (Swain et al 1990) and in vivo (Gross et al 1993; Coyle et al

1995). Furthermore, IL-4 alters the balance within macrophage populations by increasing the proportion of D1+ inductive cells at the expense of D7+ and D1/D7+ effector and suppressive cells (Tormey et al 1997).

Corticosteroids administered via the oral or inhaled route are used therapeutically in a variety of inflammatory lung diseases. In asthma, inhaled steroids reduce the number of infiltrating T cells, macrophages, dendritic cells, eosinophils and mast cells in the airway submucosa (Wang et al 1994; Burke et al 1996; Paul et al

1998). Not only is total number of lung macrophages reduced but efficacious therapy is associated with alteration in the balance between phenotypically and functionally distinct macrophage subsets (Spiteri 199, Burke et al 1996; Paul et al 1998). More specifically, in asthma there is a reduction in the proportion of D1+ inductive cells with an increase in the D7+ effector cells and D1/D7+ suppressive cells (Paul et al 1998).

There are several possible mechanisms of action of corticosteroids including suppression of pro-inflammatory cytokine release such as IL-4, IL-5, GM-CSP and RANTES (Sousa et al 1993; Wang et al 1994, 1996; Hamid & Durham 1994; Bentley

et al 1996). In vitro studies have demonstrated that glucocorticoids reduce both Th2

cytokine (IL-4 and IL-5) and to a lesser extent Thl cytokine (IPNy) production by peripheral blood mononuclear cells (Umland et al 1997). Such T cell cytokines have been shown to exert a significant effect on mature macrophages and differentiating

monocytes (Young & Hardy 1995; Ruppert et al 1991; Lee et al 1995). Thus it remains unclear whether the changes seen within the lung macrophage pool of steroid treated patients are a direct effect on monocytes/macrophages or whether they are secondary to other immunomodulating effects of steroid therapy. The present study investigates the direct effect of corticosteroids on the differentiation of peripheral blood monocytes in a controlled in vitro environment.

MATERIALS AND METHODS

Monocyte Harvest

Mononuclear cells were separated by density centrifugation (Nycomed Pharma As, Oslo, Norway) at 650 x g for 15 minutes. Mononuclear cells were washed with phosphate buffered saline (PBS) three times and suspended at a density of 1 x 10^ cells/ml in RPMI 1640 culture medium (Sigma-Aldrich Co. Ltd., Dorset, England) supplemented with 10% heat inactivated Foetal Bovine Serum, 1.25% penicillin/streptomycin and 1.25% 200mM glutamine. 2ml aliquots were then transferred to each well of 24 well culture plates. The cultures were incubated at 37^0 in 5%C0^ to separate monocytes by adherence. After 2 hours the non-adherent cells were removed by aspiration and each well was washed 3 times in PBS preheated to 37®C. 2mls of serum-fi'ee AIM V media supplemented with 2 x 10‘^M 2-mercapto ethanol was then added to each well. For each culture experiment triplicate wells were harvested at this time (= TO). The method of harvest is described below. The cell populations at TO contained consistently greater than 90% monocytes as determined by morphology; and viability (determined by trypan blue exclusion) was consistently greater than 95%. The sensitivity of this separation technique was verified by immunophenotyping with CD 14 and CD68 as described in a previous study (Tormey et al 1997). The remaining 10% of cells were predominantly B lymphocytes.

Cell culture

Adherent monocytes were cultured in 24 well plates in AIM V media (see above) for 7 days either with no addition or with the addition of fluticasone propionate (Glaxo-

Wellcome, UK). In some experiments dexamethasone [10'^ M] was used to demonstrate that the observations made were a steroid class effect and not unique to fluticasone. Dose response and time-course experiments were carried out with addition of fluticasone at different concentrations (10’*® to 10'^ M) and at different times (day 0 - day 6) during the 7 day culture period. All were added in 20|rl aliquots with control cultures receiving 20|il of sterile PBS. All solutions added were warmed to 37®C before addition. Cultures were all harvested after 7 days. At time of harvest plates were placed at 4°C for 30 minutes and then vigorously aspirated with fresh cold PBS. All cells from the wells were collected including any cells no longer adhering to the plastic substrate. Cells were counted, viability was reassessed and only cultures with a viability greater than 90% were used for analysis. Experiments to determine dose response were performed twice.

In some experiments recombinant human IL-4 (R&D Systems, Abingdon, UK) were added in 20pl aliquots to the corticosteroid-treated cell cultures on day five. Time course and dose response effect for cytokine addition were determined in chapter 2. Control cultures received 20pl sterile PBS.

Cytospin preparation

Immunocytological analysis

Cytokine ELISA

Mixed leucocyte reactions (MLR)

Statistical analysis

The effect of corticosteroids on monocyte maturation was analysed using a paired non parametric Mann Whitney test.

RESULTS

Fluticasone alters the phenotype of differentiating monocytes

With no cytokine addition, 39% of cells expressed D l, 40% of cells expressed D7 and 24% of cells expressed both Dl and D7. The addition of fluticasone reduced the proportion of D1+ cells to 9% (p < 0.05) and increased the proportion of both D7+ and D1/D7+ populations (Fig. 5.1). This effect was dose-dependent requiring a threshold concentration of fluticasone of 10'^® M with maximum effect on macrophage phenotype occurring at 10'* M (Fig. 5.2). Fluticasone exerts a similar effect on macrophage phenotype when added at different time points during monocyte differentiation (Fig. 5.3).

In vitro pharmacokinetics of fluticasone.

To demonstrate the in vitro pharmacokinetics of FP in relation to inhibitory effects on macrophage phenotype, a time course experiment was performed in which macrophage phenotype was determined at sequential time intervals following removal of FP from the culture. The inhibitory effect of FP persisted 24 hours at which time it began to wane.(Fig. 5.4).

75n JH

8

8

5 0 Î O 3 5 25H

Effect of Fluticasone Propionate on Monocyte Differentiation X Control FP Fluticasone propionate (M) D1 1 3 D7 H D 1 / D 7

Figure 5.1 Effect of fluticasone on phenotype of differentiating monocytes.

The proportions of monocytes expressing the stimulatory phenotype D1+; phagocytic phenotype D7+; and suppressive phenotype D1/D7+ after 7 days culture in the absence or presence of fluticasone propionate (FP) added on day 5. Results represent mean ± SEM..

Dose Response Effect of Fluticasone on Monocyte Differentiation 1 0 0- i2 o> u 4-*

I

o 3 Î D1/D7 Fluticasone propionate (M)

Figure 5.2 Effect of fluticasone concentration on macrophage phenotype.

Dose response graph demonstrating the effect of fluticasone (FP) concentration on the relative proportions of D1+, D7+ and D1/D7+ cells after 7 days of culture. Fluticasone addition was made on day 5. Results represent mean ± SEM.

Time course of fluticasone

addition on monocyte

differentiation

JS

8

I

0 3

q=

1

100-1 7 5 - 5 0 - 2 5 - 0 None 0 1 3 4 5 6

Time of FP addition (days)

8 4-1

Io

3 C

Î

100-1 7 5 - 5 0 - 2 5 - 0 None 0 1 3 4 5 6 D l D7 D1/D7 D1 D7 + D1/D7

Time of addition (days)

Figure 5.3 Time course for addition of fluticasone.

The effect on cell phenotype of adding FP at time 0 (after harvest) or on days 1, 3, 4, 5 or 6 of monocyte culture. Results represent the effect of cytokine addition on the relative proportions of D1+ cells, D7+ cells, and D1/D7+ cells quantified on day 7 of culture (a). In graph (b) the proportion of effector (D7) and suppressive (D1/D7) cells are grouped together. Results represent mean ± SEM of two experiments.

Duration of Fluticasone effect following

removal from culture medium

90-1 80“ 70- 60- c o 5= 40- 2 0- 1 0- 0 Nil FP(Ohr) 1 hr 3 hrs 5 hrs 7 hrs 24 hrs D1 D7 D1/D7 Time (hours)

Figure 5.4 Duration of fluticasone effect on macrophage phenotype.

Time course experiment in which the relative proportions of D1+, D7+ and D1/D7+

macrophages were determined at sequential time intervals following removal of fluticasone (FP) from the cell culture. Fluticasone addition was made on day 5. Experiment performed in duplicate. Results of typical experiment shown.

Effect of fluticasone on TNFa and TGFp production by differentiating monocytes

7 day supernatant from all cultures were tested for levels of cytokines TNFa and TGFp. Despite a trend towards decreased TGFp production induced by fluticasone, this did not reach statistical significance (Fig. 5.5). Despite the absence of a direct effect on TNFa, fluticasone inhibited the stimulatory effect of IL-4 on production of the pro-inflammatory cytokine TNFa by monocytes/macrophages (Fig. 5.6).

Modulation of monocyte differentiation by corticosteroids has functional significance

Monocytes cultured in the presence of fluticasone for 48 hours were harvested at day 7 and admixed with allogeneic peripheral blood mononuclear cells. Treatment of the monocyte stimulator population with fluticasone significantly reduced T cell proliferation by 64% (Fig. 5.7). Thus the fluticasone induced reduction in D1+ cells and increase in D7+ cells significantly inhibited the T cell stimulatory capacity of the macrophage pool.

Effect of IL-4 and Fluticasone on TGFp Production 150-1 E 1 0 0 - 2 CCL u_ O 50- 0- Control "P + IL-4

Figure 5.5 Effect of fluticasone on TGFp production

The effect of fluticasone on TGFp production by differentiating monocytes. Results represent mean ± SEM.

Effect of IL-4, IL-10 and Fluticasone

on TNFa production

1 0 0 -,

Control IL-4 FP IL4+FP

Figure 5.6 Effect of fluticasone on TNFa production.

The effect of fluticasone on TNFa production by differentiating monocytes. Results represent mean ± SEM.

T cell Proliferation

1.5n g

1

10

c 'S jS 3 0.5 (/) 0.0 " : 7

Control IL-4 FP Dex IL-4 + FP

Figure 5.7 Effect on steroid treated monocytes on T cell proliferation.

The effect of corticosteroids and IL-4 on T cell proliferation in an allogeneic MLR. The results are expressed as counts per minute of [^H] thymidine. Experiment performed 3 times, bars represent mean ± SEM of stimulation index. (Counts per minute for allogeneic reactivity without cytokine addition were reduced to unity and all other results are represented as a stimulation index in relation to this result).

The effect of dexamethasone on monocyte differentiation.

This inhibitory effect on macrophage differentiation is not unique to fluticasone but is shared by structurally different corticosteroids. The addition of dexamethasone resulted in a reduction in the proportion of D1+ cells with a concomitant increase in the proportion of cells expressing RFD7 (D7+ plus D1/D7+) (Fig. 5.8). Dexamethasone treated monocytes also inhibit T cell proliferation in an allogeneic MLR (Fig. 5.7).

Fluticasone reverses the stimulatory effect of IL-4.

Following the present investigation of the inhibitory effect of corticosteroids on normal differentiating monocytes it remained to be determined fluticasone could correct the dysregulation of monocyte development induced by IL-4. IL-4 increased the proportion of D1+ cells from 44% to 63% while reducing the proportion of cells expressing the D7+ phenotype. The concomitant addition of fluticasone reversed this effect of IL-4, with the proportion of D1+ cells decreasing from 63% to 36% and D7+ cells increasing from 35% to 62% (Fig. 5.9).Furthermore, fluticasone inhibited the increase in TNFa production induced by IL-4 (Fig. 5.6).

Monocytes cultured in the presence of fluticasone and IL-4 were harvested at day 7 and admixed with allogeneic peripheral blood mononuclear cells (PBMCs). Cells treated with IL-4 promoted a non-significant increase in allogeneic MLR stimulation index, which was inhibited by concomitant administration of fluticasone (Fig 5.7 )

60- 3 50 o § 40

L.

3 2 20 o 1 0-

Effect of dexamethasone on monocyte differentiation

D1 D7 D1/D7

Nil Dexamethasone

Figure 5.8 Effect of dexamethasone on macrophage phenotype.

Dose response graph demonstrating the effect of fluticasone (FP) concentration on the relative proportions of D1+, D7+ and D1/D7+ cells after 7 days of culture. Dexamethasone addition was made on day 5. Experiment performed in duplicate. Results of typical experiment shown.

Effect of Fluticasone and IL-4 in Combination on Monocyte Differentiation.

100n D1 ^ 75- D7 D1/D7 ■4-* c Q) 1

5.-I

3 25- Control L-4 IL-4 + FP

Figure 5.9 Effect of fluticasone and IL-4 in combination on macrophage phenotype.

The proportions of monocytes expressing the stimulatory phenotype D1+; phagocytic phenotype D7-I-; and suppressive phenotype D1/D7+ after 7 days culture in the absence or presence of fluticasone propionate (FP) and/or IL-4 added on day 5. Experiment performed in duplicate. Results of typical experiment shown.

DISCUSSION

It has previously been shown (Marianayagam & Poulter 1991) that corticosteroids alter the phenotype of mature alveolar macrophage of normal individuals. The present study extends these observations by demonstrating that contact with corticosteroids has a selective modifying effect on both the phenotype and function of differentiating monocytes. As the subsets of cells distinguished by different phenotypes have been shown to exhibit different functions (Spiteri & Poulter 1991, Spiteri et al 1992) the alteration in cell phenotype caused by steroid contact here is important in understanding the mode of action of these therapeutic agents.

The capacity for fluticasone to downregulate inductive macrophages and increase effector and suppressive macrophages is consistent with its known effects in

vivo. As well as altering the balance between macrophage subpopulations its in-situ

effects are characterised by the reduction in T cell dominated chronic peribronchial inflammation. Similarly, in the present in vitro study, changes in balance between macrophage subpopulations is accompanied by reduced antigen presentation (D1+ expression) and hence lymphocyte proliferation. These inhibitory effects are not unique to fluticasone but also shared by another glucocorticoid, dexamethasone. Similar effects of glucocorticoids in downregulating dendritic cell function in vivo

and in vitro have been described previously (Moser et al 1995).

These phenotypic and functional changes induced by fluticasone are not associated with increased TGFp production, although this cytokine is known to be a potent inhibitor of T cell function (Fargeas et al 1992; Batuman et al 1995). This is

consistent with other studies in which the inhibition of T cell proliferation by IL-10 treated monocytes was not mediated by TGFp (de Waal Maletyt et al 1991).

It is well known that steroids can effect the release of cytokines (Stosin- Grujicic & Simic 1982; Guyre et al 1988; Sousa et al 1993; Wang et al 1994, 1996; Hamid & Durham 1994; Bentley et al 1996) and may thus affect macrophage function. Corticosteroids have recently been shown to downregulate antigen induced gene expression for IL-4, IL-5, IL-13 and IFNy (Braun et al 1997) and thus potentially have a profound indirect effect on macrophage phenotype and function. However this study demonstrates that fluticasone also inhibits the capacity for IL-4 to promote differentiation of D1+ inductive macrophages. The resulting shift in balance within macrophage subpopulations towards D7+ effector and D1/D7+ suppressive cells inhibits T cell function. Thus steroids may break the cycle of inflammation regulated by IL-4. This is of particular relevance to asthma because of the increased expression of IL-4 mRNA within the bronchial wall (Ying et al 1995). Furthermore, in vitro stimulation of peripheral blood mononuclear cells by allergen

(Dermatophagoides pteronyssinus) increases IL-4 production (Leonard et al 1997b).

The present study provides an important insight into the in vitro

pharmacokinetics of fluticasone. It is effective in determining the mature macrophage phenotype when added at various time points during monocyte differentiation. This is relevant to its therapeutic efficacy because it is likely that there is a continuous turnover of recruited monocytes within the asthmatic bronchial wall. The persistence of a steroid effect for 24 hours after removal of fluticasone

from the cell culture is compatible with a high affinity for steroid receptors within monocytes/macrophages, a feature which may contribute to its potency in vivo.

Thus the present results are indirect evidence that corticosteroids may have a significant effect on those immunopathological situations where chronic inflammation is associated with dysregulation of monocyte differentiation, such as asthma (Tormey et al 1998). It also provides indirect insight into the significance of the balance between inductive and suppressive macrophages. Normally pulmonary macrophages downregulate T cell immune responses within the lower respiratory tract thus maintaining local immunological homeostasis. It has been suggested that there is a cause and effect relationship between the reduced capacity of local macrophage populations to suppress T cell activity and the chronic nature of T cell mediated inflammation (Thepen et al 1994; Poulter et al 1994). Indeed, the alveolar macrophage induced suppression of T cell induced hyperresponsiveness in asthma is reversed by allergen exposure (Spiteri et al 1994). However corticosteroids reduce chronic peribronchial inflammation while concomitantly correcting the dysregulation of monocyte differentiation. The capacity for an efficacious drug to inhibit lymphocyte proliferation by altering the balance within macrophage subpopulations is in itself indirect evidence for the importance of the macrophage in regulating inflammation in asthma.

In document La simulación de capital y el blanqueo de activos en la constitución de sociedades anónimas (página 59-63)