Cell surface antigen expression by peripheral blood monocytes in allergic asthma: results of 2.5 years therapy with inhaled beclomethasone dipropionate.

At present, inhaled glucocorticoids are widely accepted as the therapy of choice in chronic asthma. Treatment with inhaled glucocorticoids significantly suppresses local airway inflammation in asthmatics, but may also have systemic effects, e.g. a reduction of the number of circulating hypodense eosinophils or a down-modulation of HLA-DR antigen (Ag) expression by T lymphocytes in peripheral blood. However, the effect of long-term therapy with inhaled glucocorticoids on peripheral blood monocytes (PBM), which are the precursors of the most numerous cell type in the lung, the alveolar macrophage, have not yet been evaluated. We therefore investigated the expression of various cell surface Ag on PBM from non-smoking patients with allergic asthma who were treated for 2.5 years with a beta(2)-receptor agonist plus either an inhaled glucocorticoid (beclomethasone dipropionate, BDP) (n = 4) or an anticholinergic or placebo (n = 8). We compared the results with healthy volunteers (n = 7). Long-term treatment of allergic asthmatics with inhaled BDP, but not anticholinergic or placebo therapy, was associated with a significantly lower CDllb Ag expression (p < 0.04) and higher expression of CD13, CD14 and CD18 Ag (p < 0.05, p < 0.02 and p < 0.04, respectively) when compared with the healthy control subjects (n = 7). Most interestingly, PBM of asthmatics treated with inhaled BDP expressed an almost two-fold higher level of CD14 Ag on their cell surface than PBM of patients treated with anticholinergic or placebo (p < 0.03). No significant differences in the expression of CD16, CD23, CD25, CD32 and CD64 Ag or HLA-DR were observed between PBM from the different patient groups or healthy controls. Taken together, this study shows that long-term local therapy with inhaled BDP coincides with an altered expression of at least one cell surface Ag on PBM from allergic asthmatics.


Introduction
One of the major histopathological findings in asthma is chronic inflammation of the airways. In bronchial mucosal biopsies, this inflammation is characterized by an accumulation of mononuclear phagocYlte, eosinophils, mast cells and T lymphocytes.-These inflammatory cells have also been described in the bronchoalveolar lavage fluid, [5][6][7] which, in addition, has been shown to contain elevated levels of various inflammatory mediators. 7'8 It is generally believed that the airway inflammation in asthma results from the concerted actions of the different types of inflammatory cells and their prod- 362 Mediators of Inflammation Vol 5 1996 ucts, and underlies some of the clinical 1,3,5 symptoms. The most numerous cell type in both the normal and asthmatic lung is the alveolar macrophage. This cell type is nowadays known to play a central role in initiating, perpetuating, ro 9 i2 and reducing inflammatory p cesses. Monocytes and macrophages recovered from bronchoalveolar lavage fluid of asthmatics are highly activated, [13][14][15] manifested in the release of a large variety of mediators which in turn modulate the inflammatory responses of eosinophils, mast cells and T cells. 7'1 Furthermore, it has been reported that alveolar macrophages of asthmatic patients express elevated levels of (C) 1996 Rapid Science Publishers low affinity receptors for IgE (CD23), 17 which are capable of inducing cellular activation in response to specific allergen.
At present, glucocorticoids are the most effective therapy for controlling airway inflammation and clinical symptoms in chronic asthma. 18 '19 The precise mechanisms by which glucocorticoids reduce airway inflammation are not yet fully understood, but it is known that they modulate gene expression by binding to specific glucocorticoid-responsive elements in DNA. 221 One of the major mechanisms by which glucocorticoids inhibit inflammation is direct suppression of cytokine production by mononuclear phagocytes and T cells 22 or the induction of anti-inflammatory proteins such as lipocortin-1. 23 Additionally, glucocorticoids inhibit the production of pro-inflammatory mediators indirectly by suppressing the activity of several enzymes involved in the production of these mediators. Therapy with inhaled glucocorticoids for 1-4 months results in a significant decrease in the numbers of macrophages, eosinophils, mast cells, and T cells in the bronchial epithelium  and submucosa of asthmatic patients.
Only mild systemic effects have been reported with a daily dose of 1 000-2 000/g of inhaled glucocorticoids. Numbers of peripheral blood eosinophils have been shown to be reduced after treatment with inhaled glucocorticoids. 9'28 The effect of long-term treatment with inhaled glucocorticoids on peripheral blood monocytes (PBM), the precursors of alveolar macrophages, has not yet been evaluated. Identification of the effects of therapy with inhaled glucocorticoids on peripheral blood cells may be important, as increased numbers of eosinophils and activated T cells are detected in blood of asthmatic Table 1. Patient characteristics patients, in addition to the infiltration of inflammatory cells in the airways. 29 '3 In this report, we analysed the expression of various cell surface Ag by PBM of non-smoking patients with allergic asthma who were treated for 2.5 years with an inhaled 2-receptor agonist plus either inhaled beclomethasone dipropionate (BDP), or an anticholinergic or placebo. All subjects gave written informed consent.

Study design
Details of the study design have been described previously. 2 Briefly, after a double-blind randomization patients were treated with the 2receptor agonist terbutaline (two puffs of 250 pg q.i.d.) plus either: (A) inhaled glucocorticoid BDP, two puffs of 100 g q.i.d., (B) anticholinergic bronchodilator (ipratropium bromide), two puffs of 20 pg q.i.d., or (C) placebo q.i.d. At the end of the study, no significant differences with regard to FEV1 and PC20 were found between the groups receiving either ipratropium bromide or placebo. Therefore, the data of these groups were pooled for analysis as one single group (designated as anticholinergic/ placebo group). Heparinized venous blood of patients was collected after completing 2.5 years of treatment. Heparinized blood of healthy volunteers served as control.
The cell concentration was adjusted to 5 106 cells/ml.

Immunofluorescence staining
MoAb used in this study are listed in Table 2.
CDll and CD18 moAb were used because of the central role of leukocyte adhesion molecules in the recruitment of blood cells to inflammatory sites. 5 Cell surface aminopeptidase-N can be recognized by CD13 moAb, and may play a role in the inactivation of some

Results
Long-term treatment with inhaled BDP in asthma is associated with differences in the level of cell surface Ag expression by PBM The expression of various cell surface Ag by PBM from both non-smoking allergic asthmatics treated for 2.5 years with either inhaled BDP or anticholinergic/placebo, and healthy volunteers is summarized in Table 3. PBM from patients who received anticholinergic/placebo did not show any significant alterations in the levels of cell surface Ag expression when compared with PBM from healthy controls. In contrast, PBM from patients treated with inhaled BDP differed significantly in the level of expression of CD 1 l b (p < 0.04), CD13 (p < 0.05), CD14 (p < 0.02), and CD18 (p < .0.04) Ag when compared with PBM from healthy controls (Fig. 1). A reduced level of CDllb Ag expression was observed, whereas the expression of CD13, CD14, and CD18 Ag was higher. The only significant difference between the two groups of asthmatic patients concerned the level of CD14 Ag expression; PBM from patients who had received inhaled BDP expressed significantly higher levels of CD14 Ag than PBM from asthmatic patients who had received anticholinergic/placebo (Table 3, Fig. 1). Both groups of patients and the control group, however, did not differ in the relative numbers of CD14 + monocytes in peripheral blood (data not shown). The expression of RDF9 by PBM of asthmatics treated with inhaled BDP was significantly increased when compared with PBM of healthy controls (p < 0.01). However, this specific cell surface fluorescence fell within two-fold the fluorescence intensity of an isotype-matched control moAb, suggesting that monocytes express negligible levels of RFD9 Ag. No differences in the expression of CD16 (Fc,RIII), CD23 (FceRII),  CD25 (IL-2R), CD32 (Fc,RII) and CD64 (FcTRI) were observed between both groups of patients and healthy controls. The expression of CD16, CD23, and CD25 Ag fell within two-fold the background fluorescence of isotype-matched control moAb, comparable with the expression of RFD9 Ag. 366 Mediators of Inflammation Vol 5 1996 Long-term therapy with inhaled BDP and differences in subsets of peripheral blood lymphocytes In addition to analysing the cell surface Ag expression on PBM, we investigated whether long-term therapy with inhaled BDP is asso-ciated with differences in the percentage of T and B lymphocytes in peripheral blood (Table   4). No differences were observed in the percentages of either CD4 + or CD8 + T cells. However, both groups of patients had higher percentages of HLA-DR / T cells in their peripheral blood than healthy controls. Treatment with inhaled BDP did not alter the percentage of these activated T cells. Interestingly, patients who received anticholinergic/placebo, but not the patients treated with inhaled BDP, had a significantly higher percentage of B lymphocytes (CD20 + cells) in their peripheral blood compared with healthy controls (Table 4).

Discussion
In this study, we show an almost two-fold higher expression of CD14 Ag on PBM from allergic asthmatics treated for 2.5 years with an inhaled glucocorticoid (BDP, 800 tg daily) compared with patients treated with anticholinergic or placebo. In addition, long-term treatment of asthmatic patients with an inhaled glucocorticoid, but not anticholinergic or placebo, was associated with a significantly reduced expression of CD1 l b Ag on PBM, and a significantly increased expression of CD 13, CD 14, and CD 18 Ag compared with healthy control subjects.
Glucocorticoids were introduced in 1949 as a new and promising drug in the treatment of inflammatory diseases. 42 Shortly thereafter,, local administration of glucocorticoids was commenced to by-pass the unwanted systemic side effects. 43 Nowadays, inhaled glucocorticoids are widely accepted as the treatment of choice in chronic asthma. 28 Several reports showed that inhaled glucocorticoids are safe and almost free of systemic effects. However, high doses of inhaled glucocorticoids may still influence the hypothalamus-pituitary-adrenal axis. 2s'44 More evidence for systemic effects of inhaled glucocorticoids comes from studies on circulating eosinophils and T cells. Circulating hypodense eosinophils, which have been shown to exhibit a great inflammatory potential, were reduced in asthmatic subjects after short-term, treatment with inhaled glucocorticoids. 45'46 Furthermore, a small but significant reduction in HLA-DR expression by peripheral blood T cells has been reported after 6 weeks of therapy with inhaled glucocorticoids. 27 To our knowledge, modulation of PBM by inhaled glucocorticoids has not been described up till now. Leukocyte adhesion molecules, among which the CD11/CD18 family of 2 integrins, play an important role in the recruitment of cells from peripheral blood to the site of inflammation as well as in other immunological and inflammatory processes that require direct cellular interactions. 35'47'48 Both the expression and function of adhesion molecules are augmented in response to inflammatory mediators and cytokines. 49 The reduction in CD1 l b Ag expression by PBM observed here in asthmatic subjects treated with inhaled glucocorticoid, may have indirectly resulted from the local glucocorticoidmediated reduction in inflammatory mediators in the lung. Alternatively, systemically absorbed BDP may be directly responsible for the modulation of CD1 l b Ag expression. In contrast to a reduced expression of CDllb, a slightly but significantly elevated CD18 Ag expression was seen on PBM of asthmatics treated with inhaled glucocorticoid when compared with PBM of healthy control subjects. The CD18 Ag is found on the cell surface in association with one of three different chains, i.e. the CD 1 l a, CD 1 l b, or CDllc Ag. 47 Since the expression of CDlla and CD1 l c Ag were not analysed in this study, we do not know whether the observed increase in CD18 Ag expression coincides with an increased expression of CD 11 a and/or CD 11 c Ag. Long-term treatment of asthmatics with inhaled BDP was associated with an increased expression of CD13 Ag by PBM compared with healthy controls. CD13 Ag, a cell membranebound aminopeptidase-N, has been shown to play an important role in modulating the activity of regulatory oligopeptides. 6 An in- phages. This process can be inhibited by glucocorticoids. 41,52 Therefore, it may be that in this study the interference of inhaled BDP with the maturation of PBM manifests itself in the increase of CD 14 Ag expression. In this study, we also show that all asthmatic patients, treated with either inhaled glucocorticoid or anticholinergic/placebo, have significantly increased percentages of HLA-DR + T cells in their peripheral blood when compared with healthy control subjects. Long-term treatment with inhaled BDP did not reduce the percentage of circulating activated (HLA-DR+) T lymphocytes in both groups of patients. Recently, Wilson et al. 27 showed in an uncontrolled study that patients treated for 6 weeks with inhaled BDP showed a reduction in the percentage of activated T cells. Compared with our study, they used a higher dose of BDP and they did not compare their result with patients who did not receive inhaled glucocorticoids. In the present study, we also observed that patients treated with bronchodilator/placebo, but not the patients treated with inhaled BDP, had a higher percentage of B lymphocytes in peripheral blood compared with healthy controls. Therefore, treatment with inhaled glucocorticold may have influenced the proportion of circulating B lymphocytes in our group of patients.
In summary, our data demonstrate that longterm therapy with an inhaled glucocorticoid coincides with an altered expression of at least one cell surface Ag on PBM of allergic asthmatics. In the treatment of patients clinicians should be aware of these systemic effects of 368 Mediators of Inflammation Vol 5 1996 inhaled glucocorticoids. Future studies are needed to determine whether changes in cell surface Ag expression by PBM are part of the anti-inflammatory action of inhaled glucocorticoids, and whether they correlate with the glucocorticoid-induced improvement in FEV1, PC20 and clinical symptoms.