Ultraviolet B radiation and reactive oxygen species modulate interleukin-31 expression in T lymphocytes, monocytes and dendritic cells.

BACKGROUND: Interleukin (IL)-31 is a novel Th2 T-cell cytokine that induces pruritus and dermatitis in transgenic mice. While enhanced mRNA expression of this cytokine is detected in skin samples of inflammatory skin diseases, the regulation of IL-31 expression is poorly understood. OBJECTIVES: To assess the effects of ultraviolet (UV) B radiation and H2O2 on IL-31 mRNA and protein expression in skin and different peripheral blood mononuclear cells (PBMCs). METHODS: The effects of UVB radiation and H2O2, as a prototypic reactive oxygen species, on IL-31 mRNA and protein expression were analysed in various inflammation-related cells and murine skin tissue. RESULTSTreatment of cells with UVB radiation and H2 O2 strongly induced IL-31 mRNA and protein expression in human PBMCs and in the skin of SKH-1 mice. Following exposure to UVB or H2O2, we observed increased expression of IL-31 mRNA in T cells, monocytes, macrophages, and immature and especially mature dendritic cells. H2O2 treatment but not UVB radiation led to a moderate upregulation of IL-31 mRNA expression in epidermal keratinocytes and dermal fibroblasts. Pretreatment of T lymphocytes with the MAPK p38 inhibitor SB203580 or the MEK1 inhibitor U0126 reduced the stimulatory effect of H2O2. These experiments suggest that p38 is involved in the regulation of IL-31 expression in human skin. CONCLUSIONS: Our studies reveal that UVB and reactive oxygen species stimulate the expression of IL-31 in PBMCs and skin, especially in T cells, monocytes and monocyte-derived dendritic cells.

Differential regulation of the expression of transporters associated with antigen processing, TAP1 and TAP2, by cytokines and lipopolysaccharide in primary human macrophages.

OBJECTIVE: Using microarray technique we analysed global changes in gene expression of interferon-y treated primary macrophages. Among the differential expressed genes identified we focussed on the expression of the transporters associated with antigen processing, TAP1 and TAP2, which are involved in the antigen presentation via MHC class 1. Patients suffering from TAP deficiency syndrome have clinical manifestations including recurrent bacterial infections of the respiratory tract and chronic necrotizing granulomatous skin lesions. This is one reason why the regulation of TAP gene expression in antigen presenting cells such as macrophages might provide important general insights into the generation of cellular immune response to multiple pathogens. Additionally IFN-alpha is important in adjuvant tumortherapie although the working mechanisms are unknown. Because of the possibility of the TAPs to be involved in these mechanisms we studied the expression of these transporters in human macrophages after stimulation with pro-inflammatory mediators. MATERIAL AND TREATMENT: Monocyte derived macrophages were treated for 24 h with either interferon-gamma, interferon-alpha, interleukin-1 (each 100 U/ml) or lipopolysaccharide (1 microg/ml). METHODS: IFN-gamma induced gene expression was analysed using microarray technique. TAP expression was investigated by RT-PCR, northern blot- and western blot analysis. RESULTS: TAP1 and TAP2 were constitutively expressed at a low level. IFN-gamma upregulated the expression of both transporters. LPS caused an increase similar to the effect of IFN-gamma. Treatment with IFN-a stimulated also the expression, however, less than IFN-y. In contrast, IL-1beta stimulation had no effect. CONCLUSION: Our data show that the transporters associated with antigen presentation are differentially regulated by pro-inflammatory mediators in human macrophages. The finding that IFN-alpha stimulates the expression of proteins involved in cytotoxic effector functions of macrophages contributes to the understanding of the immunoregulatory role of type 1 interferons and may help to explain the efficacy of IFN-alpha in the treatment of tumors.

[Pathology of implants]. / Pathologie der Implantate.

Progress in the surgery of implants and biomaterials can be accomplished by: 1. Painstakingly analysing and registering of defaulting implants after explantation within a "National Registry of Implant Pathology". 2. Development of a DNA-microarray named "Implantat/Chronic Wound" in order to discover the differential transcriptional activities of cells brought into contact with different foreign surfaces. 3. Predictive cell-engineering combined with custom-made implant surfaces with the aim of optimal patient care.

Inflammatory response to a porcine membrane composed of fibrous collagen and elastin as dermal substitute.

The inflammatory response to a collagen/elastin membrane was studied by measuring the expression of cytokines and function associated antigens in human macrophages. Additionally the angiogenic and inflammatory activity in the chorioallantoic membrane of the chick embryo (CAM-assay) was investigated. Macrophages cultured on the membrane expressed IL-1beta mRNA as early as after 4 hours. During prolonged culturing IL-1beta mRNA levels decreased. Messenger RNA for IL-8 was detectable over the whole culture period. The anti-inflammatory cytokine IL-10 was expressed up to one day only. Phenotypic analysis revealed a decrease in the number of chronic inflammatory 25F9 positive macrophages not migrating into the membrane but a presence of these cells together with the acute inflammatory 27E10 macrophages within the membrane whereas the anti-inflammatory subtype RM3/1 was absent. In the CAM-assay the membrane stimulated angiogenesis and induced the formation of granulation tissue. Histological analysis showed that the membrane was infiltrated with macrophages, fibroblasts and endothelial cells and locally with granulocytes. These data show that the collagen/elastin membrane causes activation of macrophages, angiogenesis and the formation of inflammatory tissue. Although these processes are essential for wound healing the type of inflammation points to a chronic process which might counteract an efficient scar formation.

The chorioallantoic membrane of the chick embryo as a simple model for the study of the angiogenic and inflammatory response to biomaterials.

Angiogenesis is essential in wound healing and a common feature in chronic inflammation which is crucially involved in the biological response to biomaterials. A useful system to evaluate the angiogenic activity and the inflammatory potency of various agents is the chorioallantoic membrane (CAM) of the chick embryo. Here we examined its response to different biomaterials. Smooth materials such as PVC or the polyurethane Tecoflex either unmodified or modified by an OH- or N(CH(3))(3)(+)-end group (HEMA or MAPTAC) inhibited angiogenesis and did not induce the formation of granulation tissue. The anti-angiogenic effects of PVC, Tecoflex and its HEMA modification, however, were only seen at an early stage of development. In contrast, the MAPTAC modified Tecoflex inhibited angiogenesis over the whole time. Rough materials, e.g. filter paper or a collagen/elastin membrane, stimulated angiogenesis and induced the formation of inflammatory tissue. Histological analysis revealed that the filter material was homogeneously populated with cells consisiting mainly of macrophages, fibroblasts and endothelial cells. The collagen/elastin membrane was only partially infiltrated with cells. Among those also clusters of granulocytes were present pointing to an acute inflammatory process. These data show that the angiogenic activity and inflammatory response of biomaterials strongly depend on the chemical composition and the physical structure of the material. The CAM assay appears to be a useful tool for studying biocompatibility.

Ambroxol inhibits the release of histamine, leukotrienes and cytokines from human leukocytes and mast cells.

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.

The contact of human macrophages with extracellular matrix proteins selectively induces expression of proinflammatory cytokines.

The interaction of macrophages with proteins of the extracellular matrix (ECM) is important for the regulation of the immune and nonimmune functions displayed by these cells. Little, however, is known about the ability of different ECM proteins to transmit inflammatory signals into macrophages. Here we investigated the effect of the ECM proteins collagen type I, fibrin and fibronectin on the expression of the proinflammatory cytokines interleukin-1beta (IL-1beta) and IL-8 using RT-PCR, Northern and Western blot analysis and ELISA technique. It was found that collagen strongly induced IL-1beta and IL-8 expression in the macrophages. Fibronectin also stimulated cytokine expression, however, the amounts of the specific mRNAs were significantly lower compared to those induced by collagen. On the protein level IL-1beta revealed a close correlation to the mRNA expression. In contrast, fibrin did not elicit any IL-1beta and IL-8 response. These data show that different ECM proteins vary in their ability to induce proinflammatory cytokine expression in human macrophages suggesting that the protein composition of the ECM might be crucial in the initiation of inflammatory processes.

Generation and subcellular distribution of histamine in human blood monocytes and monocyte subsets.

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.

Cytochrome P450 1B1: a major P450 isoenzyme in human blood monocytes and macrophage subsets.

In this study, cytochrome P450 (CYP; EC 1.14.14.1)-dependent activities and P450 isoenzyme patterns were determined in human monocytes and macrophages, which play a major role in antigen processing including small molecular weight compounds which cause contact dermatitis or drug-allergic reactions. Using reverse transcriptase-polymerase chain reaction (RT-PCR) we determined the mRNA expression of eight CYPs (1A1, 1A2, 1B1, 2B6/7, 2E1, 3A3/4, 3A7 and 4B1) in human blood monocytes and macrophage subsets 27E10 and RM3/1. To study the influence of known P450 inducers, monocytes were incubated in vitro with ethanol, dexamethasone, cyclosporin A (CSA), benzanthracene (BA), phenobarbital (PB), lipopolysaccharide (LPS) and 12-O-tetradecanoyl-phorbol-13-acetat (TPA) for 24 hr. Percoll density gradient isolated monocytes as well as the pro-inflammatory macrophage subtype 27E10 expressed 1B1, 2E1 and 2B6/7. On the other hand, in the anti-inflammatory macrophage subtype RM3/1, predominantly 1B1 and to some extent 2B6/7 were found. Treatment with cyclosporin A, phenobarbital, benzanthracene or ethanol resulted in induction of the expression of 3A3/4. CYP1B1 was the predominant isoenzyme in all monocytes and macrophages. In monocytes purified by adherence or induced by benzanthracene, lipopolysaccharide or 12-O-tetradecanoyl-phorbol-13-acetat, 1A1 was also expressed. Northern blot analysis confirmed the presence of CYP1B1 in monocytes and macrophages, a presence which was also demonstrated on the protein level by immunoblot and by immunohistochemical staining of the cells. The expression of several CYPs in monocytes/macrophages suggests that these cells may be important in the metabolism of small molecular weight compounds, which play a role in allergic contact dermatitis and drug reactions. Of particular interest is the remarkably strong expression of the recently identified dioxin inducible CYP1B1, known to be present in a wide range of malignant tumors.

The effects of the glucocorticoids prednisolone, deflazacort and beclomethasone-dipropionate on the RM 3/1 macrophage in human peripheral blood.

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.

Monocyte-biomaterial interaction inducing phenotypic dynamics of monocytes: a possible role of monocyte subsets in biocompatibility.

For the in vitro study of cell-biomaterial surface interactions, the choice of cell type is crucial. In vivo data indicate that during the healing of the implant in the tissues, the pivotal cell types are the macrophages. These cells, upon interaction with any foreign material, might initiate a spectrum of responses, which could lead to acute and chronic inflammatory changes affecting the biocompatibility of the implant. Whether the mechanisms governing the type of evolving inflammatory reaction could be attributed to the macrophages functional differentiation mirrored by monocyte subsets during the polymer interaction, is poorly described. This in vitro study, therefore, attempted to investigate whether different biomaterials influence monocyte cellular activity, determined by the myeloperoxidase level and mitochondrial XTT cleavage, and phenotype dynamics characterized by the presence of CD14, RM 3/1 and 27E10 antigens. It is shown that different polymers exert differential potential to influence monocytes, both in their cellular activity and their phenotypic pattern. Thus, these findings demonstrating material-induced monocyte activation and monocyte phenotype modulation, are suggestive of the monocyte role as reporter cells in evaluating the biocompatibility of a synthetic medical device.

Influence of severe burn injury on the expression of RM 3/1 and HLA-DR antigens in human blood monocytes.

The effect of severe burns on the expression of the glucocorticoid-inducible RM 3/1 and HLA-DR antigens in blood monocytes was studied in patients with less than or more than 50% total body surface area (TBSA) burned. All patients showed a strong increase in the portion of RM 3/1+ monocytes within 1 day after injury. In patients with more than 50% TBSA, RM 3/1+ cells decreased after 2 days; in those with less than 50% TBSA, cells decreased after 3 days HLA-DR+ monocytes decreased within 4 days in both groups. In patients with less than 50% TBSA, HLA-DR+ monocytes slowly increased thereafter to basic levels. In patients with more than 50% TBSA, HLA-DR+ monocytes further decreased, then slowly increased, however, did not reach basic levels. This long-lasting decrease was evidence in the nonsurvivors. These results show that severe burns differently affect monocyte antigens. The induction of the anti-inflammatory subtype RM 3/1 and the decrease of the immunoregulatory HLA-DR antigens may contribute to the immunosuppression observed after burn injury.