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.

Immunohistochemical demonstration of migration inhibitory factor in different types of urticaria.

Because urticarial lesions can persist for extended periods of time, we have investigated the histochemical expression of an antibody against the cytokine macrophage inhibitory factor in 23 patients with different types of urticaria. Positive staining of upper and middermal dendritic cells was noted in sections from all three biopsy specimens of acute urticaria, eight of chronic urticaria, and all six of urticaria pigmentosa lesions. In all but one biopsy specimen, endothelial cells reacted as well. In three sections (two chronic urticaria, one urticaria pigmentosa), luminal lining cells of sweat glands were also noted to stain positively. In contrast, lesional skin from all eight patients with pressure urticaria was negative, as was the clinically normal skin of all patients, with the exception of one patient with urticaria pigmentosa. The data suggest that cytokines may be involved in lesions of acute type immunologic processes and that they need not be expressed in delayed type reactions.

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.

Macrophage subsets in different types of urticaria.

Biopsy specimens from lesional and uninvolved skin of nine patients with delayed pressure urticaria, three patients with acute urticaria, six patients with chronic recurrent urticaria, and four patients with urticaria pigmentosa were analyzed by routine histology and by immunochemistry for their reactivity with monoclonal antibodies to three different subsets of macrophages. Skin from 12 healthy volunteers served as control. Uninvolved skin of patients did not differ from that of healthy volunteers. An antibody against activated macrophages (27E10) was reactive to a marked extent with macrophages in wheals of pressure urticaria, more variably in acute and chronic urticaria, and practically not at all in urticaria pigmentosa. Antibodies with specificities for macrophages in healing (RM3/1) and normal (25F9) tissue reacted more markedly in all but pressure urticaria lesions, compared with normal skin. These findings indicate an active involvement of inflammatory macrophages in whealing reactions while these cells play apparently no role in cutaneous mast cell proliferation (urticaria pigmentosa).

Association of different macrophage phenotypes with infiltrating and non-infiltrating areas of tumor-host interface in colorectal carcinoma.

At the tumor-host interface (interface) of well differentiated tubulary or tubulopapillary colorectal carcinomas infiltrative, poorly demarcated and non-infiltrative, well bordered areas alternate. The composition of the inflammatory infiltrate within the desmoplastic stroma of the central tumor part and the interface was analyzed, particularly emphasizing differences between infiltrative and non-infiltrative areas of the interface. Of particular interest was the distribution of the following recently identified, functionally different human macrophage phenotypes: the 27E10-positive phenotype, an inflammatory macrophage, the 25F9-positive phenotype, a mature, resident macrophage and the RM3/1-positive phenotype, associated with anti-inflammatory function. It was found that macrophages were the dominating cells in the inflammatory infiltrate of both central tumor part and interface and that the number of B-cells and NK-cells were negligible. The 27E10-positive phenotype revealed a strong association with infiltrative areas at the interface, whereas the resident macrophage together with the RM3/1 was associated with sharply bordered tumor areas dominating within the tumor stroma, particularly in carcinomas with marked desmoplastic stroma response. These findings suggest that different macrophage phenotypes, localized in different regions of the carcinoma, have different effects on tumor cells.

In vivo effects of OK-432, inosine pranobex and its salt components on macrophage subpopulations in spleen and peritoneum of mice during the primary humoral immune response.

Subsets of macrophages (BM 8-, la- and esterase-positive subtypes) in the spleen and the peritoneum of mice were affected differently by immunomodulators during the humoral immune response. I.p. application of inosine pranobex (INPX), as well as of its salt moiety (DIPPACBA) and its dimethylaminopropanol (DIP) and acetamidobenzoic acid (PACBA) components, increased the number of nonspecific esterase-bearing cells in the peritoneum on day 3. INPX and DIPPACBA stimulated the la+ macrophage on day 3, DIPPACBA the BM 8+ macrophage only on day 1 and DIP on day 5. In spleen, DIP and PACBA had marked effects on the la+ subset in addition to a marginal effect on the BM 8+ phenotype on day 1. DIPPACBA also influenced the BM 8+ macrophage slightly on day 1. In contrast, the microbial product OK-432 stimulated BM 8+ and la+ macrophages in spleen markedly on day 1, but only marginally on days 3 and 5. However, it exerted a strong effect on both subtypes in peritoneum on days 3 and 5. OK-432 was found to be without any influence on esterase-bearing macrophages. The results show that the heterogeneity of macrophages is not only represented by subset markers, but also by their susceptibility to immunomodulators in different organs and stages of the immune response.

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.

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.

Inhibition of spontaneous and mitogen-induced lymphocyte proliferation by murine bone marrow-derived macrophages: role of prostaglandins, nitric oxides and cell-to-cell contact.

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.

Comparison of histamine release from human blood monocytes, lymphocytes, adenoidal and skin mast cells.

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.

Comparison of dermal and systemic application of glucocorticoids on the RM 3/1+ macrophage in human blood.

Dermal administration of either hydrocortisone or fluprednidene to healthy skin causes only a weak and short-lasting increase of the proportion of the anti-inflammatory macrophage RM 3/1 in the blood compared to the effect of the systemic application of glucocorticoids on this cell subtype. On the other hand, a rather permanent increase of these macrophages could be observed in untreated patients suffering from certain skin diseases, e.g. urticaria, atopic dermatitis, psoriasis.

Influence of dexamethasone on the RM 3/1-positive macrophages in the peripheral blood and tissues of a New World monkey (the marmoset Callithrix jacchus).

Subcutaneous injection of the glucocorticoid dexamethasone in the primate Callithrix jacchus increased the proportion of the macrophage subtype RM 3/1 in the blood from a basic level of 16% positive monocytes to about 65%. The elevated numbers of RM 3/1-positive cells were seen 24 and 72 h after application and were independent of the dosages used (50 or 150 micrograms/kg). Oral administration had no effect. CD4-, CD14- and CDw14-antigen expression of monocytes was not influenced by the dexamethasone treatment. In tissues, e.g. spleen, thymus, liver, skin and kidney, RM 3/1-positive macrophages revealed a similar distribution as in human tissues but no differences in glucocorticoid treated to control animals could be observed. These results show that glucocorticoids induce in the monkey Callithrix jacchus a distinct monocyte subtype similarly as in man while other macrophage phenotypes were not influenced.

Characterization of a novel anti-inflammatory factor produced by RM3/1 macrophages derived from glucocorticoid treated human monocytes.

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.

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.

Macrophages and multicellular tumor spheroids in co-culture: a three-dimensional model to study tumor-host interactions. Evidence for macrophage-mediated tumor cell proliferation and migration.

In a new co-culture model involving multicellular tumor spheroids and different phenotypes of human macrophages, we studied the effects of the latter on migration and proliferation of the human colon carcinoma cell line, HRT-18. The macrophage phenotypes are detectable with monoclonal antibodies and are inducible in culture. 12-O-tetradecanoyl-phorbol-13-acetate-activated macrophages are associated with the phenotype 27E10, which is an acute inflammatory macrophage. The glucocorticoid-induced macrophage phenotype RM3/1 is associated with the down-regulation of inflammation. The phenotype resembling the mature resident macrophage termed 25F9 arises spontaneously in prolonged culture. It could be shown that inflammatory macrophages are localized at invasive areas of the tumor-host interface of colorectal carcinoma, whereas resident and anti-inflammatory macrophages were found in the central tumor region or at well-bordered areas of the tumor-host interface. The results obtained with this co-culture model show that 27E10-associated macrophages stimulate tumor cell migration and inhibit tumor cell proliferation. RM3/1 had only a slight inhibiting effect on proliferation and a slight promoting effect on migration. The 25F9-positive macrophage-stimulated tumor cell proliferation and inhibited migration completely. This investigation indicates that this in vitro system is useful for studying different macrophage effects on tumor cells and that indeed proliferation and migration of tumor cells could be influenced in an opposite manner by different types of macrophages.

Pattern of macrophage subpopulations in post-traumatic bone infections after combined operative/antibiotic treatment.

Macrophage subpopulations were detected immunohistochemically with the aid of monoclonal antibodies in tissue sections of 15 patients with posttraumatic osteomyelitis at the beginning of therapy and after combined operative/antibiotic treatment. Macrophages represent the majority of the immunocompetent cells in osteomyelitis tissue. Before the start of therapy, the acute inflammatory macrophage subtype 27E10 was absent or rarely found in 8/13 evaluable biopsies from the osteomyelitis focus, and a further decrease in the expression of these macrophage antigens was observed after treatment. The RM3/1-positive macrophage associated with the down-regulation of inflammation was detectable to a low extent in 4/13 evaluable biopsies from the osteomyelitis focus before the start of therapy. After treatment of the infection, an increase in this subtype was found in the cellular inflammatory infiltrates in the tissue samples examined. In 8/15 biopsies a marked expression of the RM3/1 antigen was observed. At the start of treatment, the macrophage 25F9, which dominates in the late phase of inflammation, was missing in 3/13 tissue samples. After combined operative/antibiotic treatment the 25F9-positive macrophage was found in all patients, having increased in 7/14 biopsies studied. These data suggest that treatment of posttraumatic osteomyelitis leads to a local macrophage subtype distribution in the osteomyelitis focus resembling the pattern of a late inflammatory reaction.

Differential adherence of the human monocyte subsets 27E10 and RM3/1 to cytokine- or glucocorticoid-treated endothelial cells.

Monocyte-endothelial interactions are of particular importance in the regulation of inflammation. The aim of the present study was to investigate the adhesion of functional different monocyte subsets to human umbilical vein endothelial cells treated with various cytokines or a glucocorticoid. The adherence of monocyte subset 27E10, which is associated with inflammatory processes, increased after endothelial activation with interleukin-1 beta (IL-1 beta), interferon-gamma (IFN gamma), tumor necrosis factor-alpha (TNF alpha), and the glucocorticoid prednylidene (Pred). The adherence at IFN gamma-treated endothelial cells was strong after a coculture duration of 10 min with a slight increase up to 60 min. The peak value after TNF alpha stimulation was reached after 15 min, thereafter quickly decreasing. IL-1 and Pred treatment caused a maximal adherence between 15 and 30 min followed by a slow decrease. TNF alpha and particularly interleukin-6 (IL-6) enhanced the endothelial adhesion of the monocyte subtype RM3/1, which is associated with the downregulation of inflammation. The maximal adherence was found after 15 and 30 min of coculture, respectively. The results show that, through modulation of the adhesive properties of endothelial cells, cytokines and glucocorticoids affect the adherence of monocyte subsets differently. They also suggest that IL-6 plays a role in the downregulation of acute inflammation.

Macrophages and lymphocytes: alternative sources of histamine.

Some recent observations have indicated that cells other than mast cells, notably macrophages, may contain significant amounts of histamine. Using a histamine-specific radioimmunoassay, we found that human blood monocytes and lymphocytes contain about 0.05 pg histamine/cell. Various other cells, e.g. fibroblasts, colorectal tumor, kidney and ovarian cells, and murine bone marrow derived macrophages contained markedly less histamine (< 0.008 pg/cell). Ionophore A23187 (1 microM) released up to 50% of the total histamine from monocytes and lymphocytes. C5a caused a dose-dependent histamine release of up to 40% in monocytes and up to 20% in lymphocytes. Substance P induced a release only in cells of certain donors. Lipopolysaccharide, concanavalin A, and compound 48/80 had no effect. Culturing of the cells caused a loss of cellular histamine and its releasability. In view of the huge numbers of monocytes and lymphocytes in the blood, the histamine in these cells has to be taken into account under both physiological and pathophysiological conditions.

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.

[Immune reactions in chronic post-traumatic osteomyelitis. Current status determination]. / Immunreaktionen bei der chronischen posttraumatischen Osteomyelitis. Eine aktuelle Standortbestimmung.

Investigations in patients with chronic post-traumatic osteomyelitis could demonstrate several deficits in immunologic response: Phagocytic activity of phagocytes is lowered accompanied by a functional deminution of leukocyte receptors for C3. Intracellular killing is diminished. Investigations concerning T lymphocyte subpopulations verified a decrease in total T cells and helper/inducer T cells. Dysfunctions in specific humoral immune response are still debatable.