Joachim Saloga

Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma

Escape from immune surveillance is critical for tumor progression in metastatic melanoma. We assessed the function of melanoma‐derived dendritic cells (DCs) in patients presenting simultaneously with responding (rM) or progressing (pM) melanoma metastases. These rare coincidences allowed us to compare syngeneically the function of tumor DCs. CD83+ DCs were purified freshly from large responding (rDCs) or progressing (pDCs) metastases following chemo‐immunotherapy. rDCs were 5 times more potent inducers of allogeneic T‐cell proliferation than the pDCs that were used as control. Phenotypic analysis showed a marked depression of CD86 expression on pDCs. Culture supernatants from pM showed production of Th2‐type cytokines [interleukin‐10 (IL‐10)], whereas a Th1 pattern [IL‐2], interferon‐γ (IFN‐γ), IL‐12) predominated in rM. The IL‐10 detected in progressing metastases was directly derived from melanoma cells. Culture supernatants from metastases applied to DC‐supported allo‐MLR assays suppressed T‐cell responses by 50–75% in the case of pM, but not rM. Finally, in a co‐stimulation‐dependent anti‐CD3 tolerance assay, pDCs (but not rDCs) induced anergy in syngeneic CD4+ T cells. Anergy could be overcome by addition of IL‐12 or IL‐2. Our results show that melanoma‐derived factors convert DC‐antigen presenting cell function to tolerance induction against tumor tissue, changing tumor DCs to “silencers” of anti‐tumoral immune responses. Int. J. Cancer 73:309–316, 1997.

Guideline on allergen-specific immunotherapy in IgE-mediated allergic diseases

SummaryThe present guideline (S2k) on allergen-specific immunotherapy (AIT) was established by the German, Austrian and Swiss professional associations for allergy in consensus with the scientific specialist societies and professional associations in the fields of otolaryngology, dermatology and venereology, pediatric and adolescent medicine, pneumology as well as a German patient organization (German Allergy and Asthma Association; Deutscher Allergie- und Asthmabund, DAAB) according to the criteria of the Association of the Scientific Medical Societies in Germany (Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften, AWMF).AIT is a therapy with disease-modifying effects. By administering allergen extracts, specific blocking antibodies, toler-ance-inducing cells and mediators are activated. These prevent further exacerbation of the allergen-triggered immune response, block the specific immune response and attenuate the inflammatory response in tissue.Products for SCIT or SLIT cannot be compared at present due to their heterogeneous composition, nor can allergen concentrations given by different manufacturers be compared meaningfully due to the varying methods used to measure their active ingredients. Non-modified allergens are used for SCIT in the form of aqueous or physically adsorbed (depot) extracts, as well as chemically modified allergens (allergoids) as depot extracts. Allergen extracts for SLIT are used in the form of aqueous solutions or tablets.The clinical efficacy of AIT is measured using various scores as primary and secondary study endpoints. The EMA stipulates combined symptom and medication scores as primary endpoint. A harmonization of clinical endpoints, e. g., by using the combined symptom and medication scores (CSMS) recommended by the EAACI, is desirable in the future in order to permit the comparison of results from different studies. The current CONSORT recommendations from the ARIA/GA2LEN group specify standards for the evaluation, presentation and publication of study results.According to the Therapy allergen ordinance (TAV), preparations containing common allergen sources (pollen from grasses, birch, alder, hazel, house dust mites, as well as bee and wasp venom) need a marketing authorization in Germany. During the marketing authorization process, these preparations are examined regarding quality, safety and efficacy. In the opinion of the authors, authorized allergen preparations with documented efficacy and safety, or preparations tradeable under the TAV for which efficacy and safety have already been documented in clinical trials meeting WAO or EMA standards, should be preferentially used. Individual formulations (NPP) enable the prescription of rare allergen sources (e.g., pollen from ash, mugwort or ambrosia, mold Alternaria, animal allergens) for specific immunotherapy. Mixing these allergens with TAV allergens is not permitted.Allergic rhinitis and its associated co-morbidities (e. g., bronchial asthma) generate substantial direct and indirect costs. Treatment options, in particular AIT, are therefore evaluated using cost-benefit and cost-effectiveness analyses. From a long-term perspective, AIT is considered to be significantly more cost effective in allergic rhinitis and allergic asthma than pharmacotherapy, but is heavily dependent on patient compliance.Meta-analyses provide unequivocal evidence of the efficacy of SCIT and SLIT for certain allergen sources and age groups. Data from controlled studies differ in terms of scope, quality and dosing regimens and require product-specific evaluation. Therefore, evaluating individual preparations according to clearly defined criteria is recommended. A broad transfer of the efficacy of certain preparations to all preparations administered in the same way is not endorsed. The website of the German Society for Allergology and Clinical Immunology (www.dgaki.de/leitlinien/s2k-leitlinie-sit; DGAKI: Deutsche Gesellschaft für Allergologie und klinische Immunologie) provides tables with specific information on available products for AIT in Germany, Switzerland and Austria. The tables contain the number of clinical studies per product in adults and children, the year of market authorization, underlying scoring systems, number of randomized and analyzed subjects and the method of evaluation (ITT, FAS, PP), separately given for grass pollen, birch pollen and house dust mite allergens, and the status of approval for the conduct of clinical studies with these products.Strong evidence of the efficacy of SCIT in pollen allergy-induced allergic rhinoconjunctivitis in adulthood is well-documented in numerous trials and, in childhood and adolescence, in a few trials. Efficacy in house dust mite allergy is documented by a number of controlled trials in adults and few controlled trials in children. Only a few controlled trials, independent of age, are available for mold allergy (in particular Alternaria). With regard to animal dander allergies (primarily to cat allergens), only small studies, some with methodological deficiencies are available. Only a moderate and inconsistent therapeutic effect in atopic dermatitis has been observed in the quite heterogeneous studies conducted to date. SCIT has been well investigated for individual preparations in controlled bronchial asthma as defined by the Global Initiative for Asthma (GINA) 2007 and intermittent and mild persistent asthma (GINA 2005) and it is recommended as a treatment option, in addition to allergen avoidance and pharmacotherapy, provided there is a clear causal link between respiratory symptoms and the relevant allergen.The efficacy of SLIT in grass pollen-induced allergic rhinoconjunctivitis is extensively documented in adults and children, whilst its efficacy in tree pollen allergy has only been shown in adults. New controlled trials (some with high patient numbers) on house dust mite allergy provide evidence of efficacy of SLIT in adults.Compared with allergic rhinoconjunctivitis, there are only few studies on the efficacy of SLIT in allergic asthma. In this context, newer studies show an efficacy for SLIT on asthma symptoms in the subgroup of grass pollen allergic children, adolescents and adults with asthma and efficacy in primary house dust mite allergy-induced asthma in adolescents aged from 14 years and in adults.Aspects of secondary prevention, in particular the reduction of new sensitizations and reduced asthma risk, are important rationales for choosing to initiate treatment early in childhood and adolescence. In this context, those products for which the appropriate effects have been demonstrated should be considered.SCIT or SLIT with pollen or mite allergens can be performed in patients with allergic rhinoconjunctivitis using allergen extracts that have been proven to be effective in at least one double-blind placebo-controlled (DBPC) study. At present, clinical trials are underway for the indication in asthma due to house dust mite allergy, some of the results of which have already been published, whilst others are still awaited (see the DGAKI table “Approved/potentially completed studies” via www.dgaki.de/Leitlinien/s2k-Leitlinie-sit (according to www.clinicaltrialsregister.eu)). When establishing the indication for AIT, factors that favour clinical efficacy should be taken into consideration. Differences between SCIT and SLIT are to be considered primarily in terms of contraindications. In individual cases, AIT may be justifiably indicated despite the presence of contraindications.SCIT injections and the initiation of SLIT are performed by a physician experienced in this type of treatment and who is able to administer emergency treatment in the case of an allergic reaction. Patients must be fully informed about the procedure and risks of possible adverse events, and the details of this process must be documented (see “Treatment information sheet”; available as a handout via www.dgaki.de/Leitlinien/s2k-Leitlinie-sit). Treatment should be performed according to the manufacturer‘s product information leaflet. In cases where AIT is to be performed or continued by a different physician to the one who established the indication, close cooperation is required in order to ensure that treatment is implemented consistently and at low risk. In general, it is recommended that SCIT and SLIT should only be performed using preparations for which adequate proof of efficacy is available from clinical trials.Treatment adherence among AIT patients is lower than assumed by physicians, irrespective of the form of administration. Clearly, adherence is of vital importance for treatment success. Improving AIT adherence is one of the most important future goals, in order to ensure efficacy of the therapy.Severe, potentially life-threatening systemic reactions during SCIT are possible, but – providing all safety measures are adhered to – these events are very rare. Most adverse events are mild to moderate and can be treated well.Dose-dependent adverse local reactions occur frequently in the mouth and throat in SLIT. Systemic reactions have been described in SLIT, but are seen far less often than with SCIT. In terms of anaphylaxis and other severe systemic reactions, SLIT has a better safety profile than SCIT.The risk and effects of adverse systemic reactions in the setting of AIT can be effectively reduced by training of personnel, adhering to safety standards and prompt use of emergency measures, including early administration of i. m. epinephrine. Details on the acute management of anaphylactic reactions can be found in the current S2 guideline on anaphylaxis issued by the AWMF (S2-AWMF-LL Registry Number 061-025).AIT is undergoing some innovative developments in many areas (e. g., allergen characterization, new administration routes, adjuvants, faster and safer dose escalation protocols), some of which are already being investigated in clinical trials.Cite this as Pfaar O, Bacher

Identification and induction of human keratinocyte-derived IL-12.

Interleukin 12 is a heterodimeric molecule that serves as a potent co-stimulator enhancing the development of Th1 cells. As one of the classical Th1 cell-mediated responses is contact sensitivity in skin, we wondered whether IL-12 might be produced by epidermal cells and serve as a mediator of this immune response. Using a sensitive, quantitative PCR technique we demonstrate that p35 chain mRNA of IL-12 is produced constitutively by human epidermal cells, whereas p40 chain mRNA can only be detected in epidermis treated with contact allergen, but not epidermis exposed to irritants or tolerogens. Time course studies showed a dramatic induction of IL-12 p40 mRNA 4 h after in vivo allergen treatment reaching peak strength after 6 h. In cell depletion assays we show that epidermal keratinocytes are the major source of this cytokine in the epidermis. This was further supported by analysis of mRNA derived from the human keratinocyte cell line HaCat expressing IL-12 p35 and p40 mRNA upon stimulation. The presence of bioactive IL-12 in supernatants derived from allergen-stimulated epidermal cells was demonstrated by IL-12-specific bioassay. Additional evidence for the functional importance of IL-12 in primary immune reactions in skin was obtained in allogeneic proliferation assays using human haptenated epidermal cells containing Langerhans cells as APC and allogeneic CD4+ T cells as responders. Anti-IL-12 mAb inhibited the proliferation of T cells by approximately 50%. In aggregate our data demonstrate that nonlymphoid keratinocytes are capable of producing functional IL-12 and provide evidence for the functional significance of IL-12 in primary immune responses in skin.

Decreased release of histamine and sulfidoleukotrienes by human peripheral blood leukocytes after wasp venom immunotherapy is partially due to induction of IL-10 and IFN-γ production of T cells

BACKGROUND Recent studies provide evidence that venom immunotherapy (VIT) alters the pattern of cytokine production by inducing an allergen-specific T-cell shift in cytokine expression from TH2 (IL-4, IL-5) to TH1 (IFN-gamma) cytokines and also inducing the production of IL-10. OBJECTIVE This study was carried out to analyze whether these changes in cytokine production of T cells already observed 1 week after the initiation of VIT in subjects with wasp venom allergy also influence the reactivity of effector cells, such as mast cells and basophils. METHODS All subjects included in this study had a history of severe systemic allergic reactions to wasp stings and positive skin test responses with venom and venom-specific IgE in the sera. Peripheral blood leukocytes were isolated before and after the initiation of VIT (rush therapy reaching a maintenance dose of 100 microg venom injected subcutaneously within 1 week) and preincubated with or without addition of IL-10, IFN-gamma, IL-10 + IFN-gamma, anti-IL-10, or anti-IFN-gamma. After stimulation with wasp venom, histamine and sulfidoleukotriene release were assessed by ELISA and compared with spontaneous release and total histamine content. RESULTS After the induction of VIT, venom-induced absolute and relative histamine and sulfidoleukotriene release were reduced. This was at least partially due to the induction of IFN-gamma and IL-10 production, because (1) neutralization of IL-10 and IFN-gamma by mAbs partially restored the release after the initiation of VIT and (2) the addition of exogenous IFN-gamma and IL-10 caused a statistically significant diminution of the venom-induced histamine and sulfidoleukotriene release before VIT. Depletion of CD2(+) T cells also restored the releasability after VIT. CONCLUSION These data indicate that T cells (producing IL-10 and IFN-gamma after VIT) play a key role for the inhibition of histamine and sulfidoleukotriene release of effector cells.

Development and transfer of immediate cutaneous hypersensitivity in mice exposed to aerosolized antigen.

We previously showed that BALB/c mice sensitized to ovalbumin (OVA) by brief daily inhalations of antigen over 10 consecutive days exhibit elevated antigen-specific serum IgE antibody levels and increased airways responsiveness. For the first time, we now show that animals sensitized in this fashion to either OVA or ragweed (RGW) develop immediate hypersensitivity skin test reactions when challenged 2 d after completion of the sensitization protocol. Skin testing, performed by direct assessment of wheal formation after intradermal injection of allergen, was sensitive and specific, since animals exposed to RGW by inhalation only responded to RGW, and OVA-sensitized animals responded only to OVA. Positive reactions were associated with mast cell degranulation, whereas control injections were not. Since only sensitized IgE high responder BALB/c mice but neither nonsensitized BALB/c mice nor OVA-sensitized IgE low responder SJL/J mice exhibited wheal responses, induction of OVA-specific IgE appeared to be essential for the mediation of OVA-specific immediate hypersensitivity reactions of the skin in this model. Passive cutaneous anaphylaxis (PCA) testing confirmed the presence of antigen-specific IgE in the serum. Mice that developed IgG (predominantly IgG2b) anti-OVA antibodies did not respond to OVA injection, indicating that OVA-specific IgG was not involved in this system. Further support for the role of IgE in the immediate hypersensitivity response included the wheal response to intradermal injection of anti-IgE antibody that occurred in OVA- and RGW-sensitized mice at 10-fold lower concentrations than in nonsensitized BALB/c mice and not in sensitized SJL/J mice. After transfer of mononuclear cells from peribronchial lymph nodes of OVA- or RGW-sensitized BALB/c mice, naive, syngeneic recipients developed antigen-specific IgE and specific immediate hypersensitivity responses, indicating that the local lymphoid tissue at the site of sensitization can transfer responsiveness to these allergens. These results demonstrate for the first time the ability to elicit and study IgE-mediated immediate skin hypersensitivity responses in the mouse and illustrate the association of increased antigen-specific and total serum IgE levels, airways hyperresponsiveness, and antigen-specific immediate cutaneous reactivity after sensitization to allergen via the airways.

Effects of glycation of the model food allergen ovalbumin on antigen uptake and presentation by human dendritic cells

Advanced glycation endproducts (AGEs) of food proteins resulting from the Maillard reaction after cooking or heating may have particular importance in food allergy. The underlying immunological mechanisms are only poorly understood. The aim of the study was to examine the effects of AGE derived from the model food allergen ovalbumin (AGE‐OVA) on dendritic cells (DCs), their immunostimulatory capacity and the T‐cell response compared with regular OVA. For this purpose, human immature DCs were exposed to fluorescein isothiocyanate (FITC)‐labelled AGE‐OVA and FITC‐labelled regular OVA and uptake was analysed by flow cytometry and fluorescence microscopy. Furthermore, autologous CD4+ T‐cell proliferation and cytokine production induced by mature DCs loaded with AGE‐OVA were compared with those induced by mature DCs loaded with OVA. Finally, expression of the receptor for advanced glycation endproducts (RAGE) and activation of the transcription factor nuclear factor (NF)‐κB by AGE were investigated. Internalization of FITC‐AGE‐OVA by immature DCs was significantly increased compared with FITC‐OVA. Blocking the mannose receptor, macropinocytosis or the scavenger receptor strongly reduced uptake of both FITC‐OVA and FITC‐AGE‐OVA. In a comparison of CD4+ T cells co‐cultured with AGE‐OVA‐loaded mature DCs versus those co‐cultured with OVA‐loaded mature DCs, AGE‐OVA DCs were found to produce more interleukin (IL)‐6 and to induce a stronger T helper type 2 (Th2) and a weaker Th1 cytokine response, while there was no difference in proliferation of CD4+ T cells. The expression of RAGE was higher on immature DCs compared with mature DCs. AGE‐OVA‐exposed immature DCs showed a stronger expression of RAGE and activation of the transcription factor NF‐κB compared with OVA‐loaded immature DCs. Our data indicate that AGE‐OVA may be more immunogenic/allergenic than regular OVA.

Increased acetylcholine levels in skin biopsies of patients with atopic dermatitis.

Recent experimental evidence indicates that non-neuronal acetylcholine is involved in the regulation of basic cell functions. Here we investigated the cholinergic system in the skin of healthy volunteers and patients with atopic dermatitis (AD). The synthesizing enzyme, choline-acetyltransferase (ChAT), was studied by anti-ChAT immunohistochemistry and enzyme assay. Skin biopsies taken from healthy volunteers and from AD patients were separated into the 2 mm superfical (epidermis and upper dermis) and 3 mm underlying portion (deeper dermis and subcutis). ChAT enzyme activity was detected in homogenized skin and subcutaneous fat (about 13 nmol/mg protein/h). ChAT immunoreactivity was expressed in keratinocytes, hair papilla, sebaceous and eccrine sweat glands, endothelial cells and mast cells. In healthy volunteers the superficial and underlying portion of skin biopsies contained 130 +/- 30 and 550 +/- 170 pmol/g acetylcholine (n = 12), respectively. In AD patients (n = 7) acetylcholine was increased 14-fold in the superficial and 3-fold in the underlying biopsy portion. The present study demonstrates the widespread expression of ChAT protein in the vast majority of human skin cells. Tissue levels of acetylcholine are greatly (14-fold) enhanced in the superficial 2 mm skin of AD patients.

Die spezifische Immuntherapie (Hyposensibilisierung) bei IgE-vermittelten allergischen Erkrankungen

The present guideline on allergen-specific immunotherapy (SIT) was established by the German allergy societies in conjunction with other scientific and medical societies (dermatology, ear-nose-throat, pediatrics, lung and airway diseases) and a patient support group according to criteria of the Association of the Scientific Medical Societies in Germany (AWMF). Subcutaneous immunotherapy (SCIT) is a unique causal treatment of IgE-mediated allergic diseases and induces longterm tolerance to the applied allergens due to numerous immunologic effects. Non-modified allergens are used as aqueous or physically coupled (depot) allergen extracts, chemically modified allergens (allergoids) are used as depot extracts for SCIT. Efficacy of SCIT has been demonstrated for pollen and house dust mite allergens in a large number of studies in patients with allergic rhinoconjunctivitis, and for animal dander (cat) and mold allergens (Alternaria, Cladosporium) in few studies. SCIT has been well-studied in intermittent and mild persisting IgE-mediated allergic asthma and is recommended as a therapeutic option besides allergen avoidance and pharmacotherapy. Preventive aspects, particularly reduced development of bronchial asthma and less novel allergic sensitizations, are increasingly considered during the decision for SCIT. In case of systemic reactions due to Hymenoptera (bee, wasp) venom allergy SCIT has excellent efficacy and should be continued for at least 3 - 5 years. An extended, sometimes lifelong SCIT, is necessary in a few patients. SCIT is indicated in patients with IgE-mediated sensitizations and corresponding clinical symptoms to allergens which do not permit allergen avoidance and which are available as suitable extracts. Diagnostic procedures, indication and selection of appropriate allergens for SCIT are made by a physician with certified training or qualified knowledge and skills in allergology. Contraindications have to be considered on an individual basis. Injections of SCIT are administered by a physician experienced in this therapy and who is able to perform emergency treatment in case of an allergic adverse event. Patients information and documentation are mandatory previous to the start of SCIT. Children tolerate SCIT very well and benefit especially from its immuno-modulatory effects. Systemic adverse reactions can occur due to SCIT, being rare in case of complete adherence to safety standards. Most adverse events are mild to moderate and easily treatable. Risk factors for and sequels of unwanted systemic effects can effectively be minimized by training the staff members involved, adhering to safety standards and immediate emergency treatment. Sublingual immunotherapy (SLIT) is an option for adults with allergic rhinoconjunctivitis due to pollen allergens, particularly if SCIT is not suitable. In house dust mite allergy and allergic asthma, SLIT does not substitute SCIT. Due to the present data, SLIT is not recommended for routine use in children and adolescents. A final conclusion on SLIT for children and adolescents is warranted if further study results are available. Various research fields like allergen characterization, routes of application, adjuvants, updosing regimen and preventive aspects demonstrate new developments in SIT being currently examined for clinical efficacy.

Influence of extracellular matrix proteins on the development of cultured human dendritic cells

The development of dendritic cells (DC) is still only partly understood. Recently established culture systems using CD34+ cells or monocytes as precursor cells for the generation of DC indicate the necessity of pro‐inflammatory cytokines for their development. In vivo the contact to other cells or to the proteins of the extracellular matrix might also be essential for their development. In our experiments we used granulocyte‐macrophage colony‐stimulating factor‐ and IL‐4‐treated human monocytes as precursor cells to investigate the interaction of DC at different maturation stages with the matrix proteins fibronectin, collagen type I and collagen type IV. We demonstrate a strong β1‐integrin‐mediated adherence of immature DC to fibronectin that is lost completely during maturation. The binding to collagen type I was less strong but induced a maturation of the precursor cells. After 3 days of culture on this protein, the cells showed all features of fully matured DC such as expression of CD83 and an excellent allostimulatory capacity. The reason for this effect was shown to be the induction of TNF‐α production by the DC themselves. In contrast to the adhesion to fibronectin, the maturation and the cytokine production of DC induced by collagen type I could not be inhibited by blocking of β1‐integrins. These results indicate that proteins of the extracellular matrix play an important role in the development and function of human DC.

The Role of Interleukin 10 in the Regulation of Allergic Immune Responses

Several clinical studies and animal models have shown that Th2 lymphocytes play a key role in the pathophysiology of IgE-mediated allergic immune responses like allergic rhinitis and asthma or venom anaphylaxis. Classical specific immunotherapy (SIT) that has been proven to be clinically effective can serve as a role model for immunological changes that are associated with amelioration of allergic diseases. During SIT, the Th2-dominated immune response is modified towards a Th1 response leading to a decline in allergen-specific IgE and an increase in allergen-specific IgG production. Most importantly, however, production of the immunosuppressive/-regulatory cytokine interleukin 10 (IL-10) is also induced leading to T cell tolerance and prevention of tissue inflammation. In this article the role of IL-10-producing T cells in the regulation of allergic immune responses will be discussed.