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OBJECTIVES AND DESIGN: The effects of the mucolytic agents ambroxol and N-acetylcystein (NAC) were studied on the release of histamine, leukotrienes, cytokines and superoxide anions from a variety of cells involved in the pathogenesis of allergic inflammation. SUBJECTS: Mast cells were isolated from human adenoids and skin (n = 5-6). Basophils, monocytes and granulocytes were obtained from Buffy-coat blood obtained from healthy blood donors (n = 4-7) and enriched by density centrifugation. TREATMENT AND METHODS: Ambroxol or NAC were added to the cells for different periods before stimulation with various immunological and non-immunological secretagogues. Histamine release from mast cells, basophils and monocytes was assayed either by radioimmunoassay or spectrofluorometrically. LTC4 (basophils), LTB4 (neutrophil/eosinophil granulocytes or monocytes), IL-4 and IL-13 (basophils) were measured by ELISA. RESULTS: Ambroxol inhibited histamine release by more than 50% from human adenoidal mast cells (1000 microM ambroxol) and skin mast cells (100 microM ambroxol) stimulated by Con A and compound 48/80, respectively. Ambroxol (100 microM) strikingly inhibited anti-IgE induced release of both histamine, LTC4, IL-4 and IL-13 from basophils and reduced both histamine and LTB4 release induced by C5a or Zymosan in monocytes. The drug also reduced LTB4 and superoxide anion production in granulocytes stimulated by zymosan or fMLP. In all cell types studied, ambroxol was more efficacious following a short preincubation (5-15 min) of the drug with the cells before stimulation. In contrast, NAC produced no clear effects on any of the different cell types studied, regardless of the preincubation period, the concentration or the stimulus employed. CONCLUSIONS: Unlike NAC, ambroxol is able to not only inhibit acute mediator release from mast cells and leukocytes but also reduce immunomodulatory cytokine generation from basophils and may have beneficial effects in the treatment of allergic respiratory diseases.

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OBJECTIVE AND DESIGN: This study was designed to establish the sites of formation and storage of histamine and histidine decarboxylase (HDC) in human monocytes and two of their subsets. MATERIALS AND METHODS: The experiments were carried out using monocytes from buffy coats of healthy blood donors. Histamine was quantitated by RIA, HDC activity by the formation of histamine. RESULTS: The monocyte subtype RM3/1 contained significantly more histamine than the subset 27E10 (0.041+/-0.025 vs. 0.005+/-0.004 pg/cell, p < 0.05) and also more HDC activity and HDC mRNA. After fractionation of monocyte homogenates in a discontinuous Percoll gradient or by differential centrifugation more than 80% of both, HDC activity and histamine, were recovered from the cytosolic fractions. About 50% of this histamine was found to be bound to proteins. CONCLUSIONS: In monocytes histamine and HDC are colocalized in the cytoplasm indicating a subcellular distribution different from mast cells or basophils. The data also show that histamine is synthesized by the monocytes themselves.

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Different glucocorticoids (GC) applied intravenously, subcutaneously or in vitro exert only small differences in the ability to raise RM 3/1 macrophages from human blood monocytes. Dermal application however reveals a dose-dependent difference between GC. The present experiments were designed to study the efficacy of oral and topical application in this model. We also intended to obtain some information about the qualitative differences between the effects of prednisolone and deflazacort, which was reported to cause less adverse reactions than other GC. Both GC were orally administered to probands in doses regarded as equivalent with respect to general GC effects by the manufacturers of deflazacort (5-6 mg or multiples). A single dose of 5 mg prednisolone had no effect; 50 mg increased the number of RM 3/1 macrophages within 12 h from a basal level of 8.5% to about 80% similar to an intravenous or subcutaneous administration of GC. 10 mg prednisolone enhanced the number of RM 3/1 macrophages also within 12 h, reaching a mean maximum of about 60% at 24 h, declining thereafter. 12 mg deflazacort raised the number of RM 3/1 macrophages much slower, reaching a maximum of 30% (average) after 48 h. The interindividual variation was found to be mainly the time lag between dosage and maximum effect. Interindividual differences of prednisolone effects concerned mainly the maximal increase of RM 3/1 macrophages after 24 h. These results show that in this test system deflazacort was found to be less effective than expected. To elucidate the topical influence of GC, probands inhaled twice daily 0.5 mg beclomethasone-dipropionate over a period of 11 days. No effect on the number of RM 3/1 macrophages was observed, suggesting that beclomethasone applied in this dose did not cause systemic GC reactions.

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Glucocorticoids exert their anti-inflammatory activity by modulating the functions of various cell types including macrophages. They also induce the generation of a distinct macrophage subtype defined by the surface antigen RM3/1 which appears to be associated with the down-regulation of inflammation. Supernatants from these cells were found to exert a dose-dependent anti-inflammatory effect, particularly in the early phase as shown in the 5-hydroxytryptamine (5-HT) induced footpad edema of mice. By using conventional purification methods the anti-inflammatory factor was found to have a molecular mass of about 78 kD and an isoelectric point of about 7.9. Heat lability and sensitivity to trypsin and proteinase K indicate the protein nature of the anti-inflammatory factor. The inhibition of the early phase of inflammation and the molecular weight suggest that the anti-inflammatory agent released from RM3/1 macrophages is a novel protein different from other anti-inflammatory proteins described so far.

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Monocytes and lymphocytes from human blood contain 0.043 +/- 0.007 and 0.053 +/- 0.014 pg histamine/cell, respectively, which can be released by a number of stimulants (A 23187, C5a, substance P, specific allergen). The release process takes 60-120 min to reach its end point, in contrast to tissue mast cells which complete the release within 1-3 min. Both, ketotifen (10(-7) - 10(-5) M) and disodium cromoglycate (10(-5) - 10(-3) M) inhibited histamine release dose dependently up to 40-45%, which might be particularly relevant during the later stages of acute allergic or pseudoallergic reactions.

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Macrophage subtypes were detected in cryostat sections of biopsies from patients with chronic osteomyelitis, acute joint infections and normal bone marrow, using monoclonal antibodies against different macrophage populations. The resident macrophage subtype 25F9, the gluco-corticoid-inducible macrophage RM 3/1 and the inflammatory type 27E10 were found in abundance in acute infections. They were also present in tissue sections of uninflamed bone marrow. By contrast, in about 50% of the biopsies from patients with chronic osteomyelitis a reduced number of macrophage subtypes, or even the lack of one or more macrophage subpopulations was found. The unusual absence of macrophage phenotypes seems to be restricted to the area of osteomyelitis because in the tissues of inflamed sinuses in these patients, the macrophage subtypes were present. These findings suggest a disturbance at the level of the macrophages which may contribute to the persistence of the inflammatory process in osteomyelitis.

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Spontaneous and mitogen-stimulated proliferation of spleen lymphocytes was inhibited by coculturing the cells with murine bone marrow derived macrophages (BM-M phi). The inhibitory effect was found to be dependent on the maturation stage of BM-M phi reflected by the appearance of the phenotype BM 8 and on the number of BM-M phi added to the lymphocytes. The spontaneous proliferation was only inhibited by the addition of high numbers of BM-M phi of late maturation stage. The lipopolysaccharide (Lps) induced response was strongly suppressed by increasing proportions of BM-M phi of either cultivation stage. The suppressive effect on the concanavalin A (Con A)-induced proliferative activity increased with the maturation of the macrophages. Inhibition of prostanoid release with indomethacin had no effect. Blocking of nitric oxide synthesis partially reversed the inhibitory effect of macrophages mainly on the Con-A response. Addition of culture supernatant of BM-M phi to the lymphocytes did not mimic the effect of the macrophages themselves. BM-M phi only slightly inhibited the proliferation when lymphocyte-BM-M phi cell-to-cell contact was prevented. These results show that BM-M phi differently influence the spontaneous and mitogen-induced lymphoproliferation and that the inhibitory effect of BM-M phi on the lymphocyte proliferation is only partially mediated by secretory products of macrophages but apparently requires cell-to-cell contact between both cell types.

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