Volker Blaschke

Rapid quantitation of proinflammatory and chemoattractant cytokine expression in small tissue samples and monocyte-derived dendritic cells: validation of a new real-time RT-PCR technology

The analysis of cytokine profiles plays a central part in the characterization of disease-related inflammatory pathways and the identification of functional properties of immune cell subpopulations. Because tissue biopsy samples are too small to allow the detection of cytokine protein, the detection of mRNA by RT-PCR analysis is often used to investigate the cytokine milieu in inflammatory lesions. RT-PCR itself is a qualitative method, indicating the presence or absence of specific transcripts. With the use of internal or external standards it may also serve as a quantitative method. The most widely accepted method is quantitative competitive RT-PCR, based on internal shortened standards. Recently, online real-time PCR has been introduced (LightCycler), which allows quantitation in less than 30 min. Here, we have tested its use for the analysis of cytokine gene expression in different experimental in vitro and ex vivo settings. First, we compared quantitative competitive RT-PCR with real-time RT-PCR in the quantitation of transcription levels of the CD4(+) cell-specific chemoattractant Interleukin-16 during the maturation of monocyte-derived dendritic cells, and found a good correlation between both methods. Second, differences in the amounts of IL-16 mRNA in synovial tissue from patients with rheumatoid arthritis and osteoarthritis as assessed by real-time RT-PCR paralleled differences in the level of IL-16 protein in the synovial fluid. Finally, we employed real-time RT-PCR to study the cutaneous expression of several cytokines during experimental immunomodulatory therapy of psoriasis by Interleukin-10, and demonstrate that the technique is suitable for pharmacogenomic monitoring. In summary, real-time RT-PCR is a sensitive and rapid tool for quantifying mRNA expression even with small quantities of tissue. The results obtained do not differ from those generated by quantitative competitive RT-PCR.

Engagement of the FcεRI Stimulates the Production of IL-16 in Langerhans Cell-Like Dendritic Cells

Preferential uptake and presentation of IgE-bound allergens by epidermal Langerhans cells (LC) via the high affinity IgE receptor, FcεRI, is regarded as an important mechanism in the induction of cutaneous inflammation in atopic dermatitis. Here, we show that activation of monocyte-derived LC-like dendritic cells (LLDC) through engagement of FcεRI induces the expression of IL-16, a chemoattractant factor for dendritic cells, CD4+ T cells, and eosinophils. We found that ligation of FcεRI on LLDC derived from atopic dermatitis patients that express high levels of FcεRI increases IL-16 mRNA expression and storage of intracellular IL-16 protein and enhances the secretion of mature IL-16 in a biphasic manner. An early release of IL-16 (peak at 4 h) is independent of protein synthesis, while a more delayed release (peak at 12 h) requires protein synthesis and occurs subsequent to the induction of IL-16 mRNA and intracellular accumulation of pro-IL-16. There was evidence that LLDC use caspase-1 to process IL-16, as inhibition of caspase-1, but not of caspase-3, partially prevented the release of IL-16 in response to ligation of FcεRI. In an in vivo model of IgE-dependent LC activation, the atopy patch test, positive skin reactions were also associated with the induction of IL-16 in epidermal dendritic cells. These data indicate that IL-16 released from LC after allergen-mediated activation through FcεRI may link IgE-driven and cellular inflammatory responses in diseases such as atopic dermatitis.

Expression of the T-cell chemoattractant chemokine lymphotactin in Crohn's disease.

Recruitment of lymphocytes is a prominent feature of the inflammatory process in Crohns disease (CD). The present study was undertaken to investigate the expression of the novel lymphocyte-specific chemoattractant lymphotactin (Lptn) as a potential regulatory factor for the recruitment of T cells in CD. The expression of Lptn mRNA was quantified in resection specimens of patients with CD in comparison to normal controls without signs of inflammation by real-time quantitative reverse transcriptase-polymerase chain reaction and localized by nonradioactive in situ hybridization. Furthermore, the phenotype of cells expressing Lptn mRNA was characterized. In contrast to normal controls Lptn mRNA was significantly increased in tissue samples affected by CD. Cells expressing Lptn were identified as T cells, mast cells, and unexpectedly dendritic cells. Lptn mRNA was found to be up-regulated on stimulation with phorbol-12-myristate-13-acetate and concanavalin A in T cells isolated from peripheral blood, which could be prevented by dexamethasone, cyclosporine A, and FK506. A similar regulation mechanism could be identified for the Lptn receptor GPR-5 in peripheral T cells. In addition, Lptn mRNA expression could be induced in mature monocyte-derived dendritic cells. The results indicate that local expression of Lptn by activated T cells and to a lesser extent by mast cells and dendritic cells represents a key regulator for lymphocyte trafficking and maintenance of the inflammatory process observed in CD, which might be partly mediated through an autocrine/paracrine pathway of activated T cells.

Exclusive expression of the Gs-linked prostaglandin E2 receptor subtype 4 mRNA in human mononuclear Jurkat and KM-3 cells and coexpression of subtype 4 and 2 mRNA in U-937 cells

Prostaglandin E2 (PGE2) is regarded as a potent regulator of the immune system. It can regulate apoptosis in monunuclear cells and modulate the cytokine secretion pattern from T‐helper cell subpopulations via an increase in cyclic AMP (cAMP). Of the 4 PGE2 receptor subtypes (EP1–EP4) that are defined pharmacologically by their affinity to subtype‐specific ligands and their coupling to G proteins, EP2 and EP4 receptors couple to Gs. It is as yet unknown which of these two receptor subtypes mediates the immunomodulatory effects. By quantitative RT‐PCR, the mRNA for EP4 receptors was demonstrated and quantified in the human mononuclear cell lines Jurkat, KM‐3 and U‐937. However, EP2 receptor mRNA was only present in U‐937 cells and was 100‐fold less abundant than EP4 receptor mRNA. PGE2 increased cAMP formation with an ED50 of 50–100 nM in all cell lines. cAMP formation was inhibited by the EP4R‐specific antagonist AH23848. Since AH23848 inhibited PGE2‐induced cAMP formation in U‐937 cells to a similar extent as in Jurkat and KM‐3, EP2 receptors seem to play, if any, only a secondary role for the PGE2‐mediated cAMP formation in U‐937 cells.

THE C-TERMINAL DOMAIN OF THE GS-COUPLED EP4 RECEPTOR CONFERS AGONIST-DEPENDENT COUPLING CONTROL TO GI BUT NO COUPLING TO GS IN A RECEPTOR HYBRID WITH THE GI-COUPLED EP3 RECEPTOR

Prostaglandin E2 receptors (EPR) belong to the family of G‐protein‐coupled receptors with 7 transmembrane domains. They form a family of four subtypes, which are linked to different G‐proteins. EP1R are coupled to Gq, EP2 and EP4R to Gs and EP3R to Gi. Different C‐terminal splice variants of the bovine EP3R are coupled to different G‐proteins. A mouse EP3R whose C‐terminal domain had been partially truncated no longer showed agonist‐induced Gi‐protein activation and was constitutively active. In order to test the hypothesis that the C‐terminal domain confers coupling specificity of the receptors on the respective G‐proteins, a cDNA for a hybrid rEP3hEP4R, containing the N‐terminal main portion of the Gi‐coupled rat EP3βR including the 7th transmembrane domain and the intracellular C‐terminal domain of the Gs‐coupled human EP4R, was generated by PCR. HEK293 cells transiently transfected with the chimeric rEP3hEP4R cDNA expressed a plasma membrane PGE2 binding site with a slightly lower K d value for PGE2 but an identical binding profile for receptor‐specific ligands as cells transfected with the native rat EP3βR. In HepG2 cells stably transfected with the chimeric rEP3hEP4R cDNA PGE2 did not increase cAMP formation characteristic of Gs coupling but attenuated the forskolin‐stimulated cAMP synthesis characteristic of Gi coupling. This effect was inhibited by pre‐treatment of the cells with pertussis toxin. Thus, the hybrid receptor behaved both in binding and in functional coupling characteristics as the native rat EP3βR. Apparently, the intracellular C‐terminal domain did not confer coupling specificity but coupling control, i.e. allowed a signalling state of the receptor only with agonist binding.

The maturation-dependent production of interleukin-16 is impaired in monocyte-derived dendritic cells from atopic dermatitis patients but is restored by inflammatory cytokines TNF-α and IL-1β

Background:  Maturation of dendritic cells (DCs) influences important DC functions such as production of cytokines. Recently, DCs were identified as a source of interleukin‐16 (IL‐16), a chemotactic factor for DCs themselves, CD4+ T cells, and eosinophils. There is evidence that DC‐derived IL‐16 may contribute to the pathogenesis of atopic dermatitis (AD).

Periorbital allergic contact dermatitis from oxybuprocaine

Keywords: allergic contact dermatitis; oxybuprocaine; local anesthetics; eyedrops; ophthalmics; medicaments; elderly patients; periorbital skin; lack of cross-sensitivity