Thymic stromal lymphopoietin production induced by skin irritation results from concomitant activation of protease-activated receptor 2 and interleukin 1 pathways.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) mediates proallergic T helper 2-type responses by acting on leucocytes. Endogenous pathways regulating TSLP production are poorly defined. OBJECTIVES: To uncover the mechanisms by which skin barrier disruption elicits TSLP production and to delineate the level at which individual mechanistic components may converge. METHODS: A combination of primary keratinocytes, skin explants and in vivo strategies was employed. Murine skin was tape stripped in the presence of neutralizing antibodies or antagonists. Cells and explants were stimulated with interleukin (IL)-1 and protease-activated receptor 2 agonist (PAR-2-Ag). TSLP levels were quantified by enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction. Chromatin immunoprecipitation and promoter reporter assays were used to examine recruitment and functional activity of nuclear factor kappa B (NF-κB) at the TSLP promoter. RESULTS: TSLP induction in mouse skin occurred in a PAR-2- and IL-1-dependent manner. This scenario was duplicated by exogenous IL-1 plus PAR-2-Ag vs. each stimulus alone. Joint activity of PAR-2 and IL-1 was also observed in human keratinocytes. The TSLP promoter was identified as the target of PAR-2/IL-1, whereby PAR-2 activation augmented the recruitment of NF-κB and transcriptional activation over IL-1 alone. Combined treatment showed activity at concentrations of IL-1 unable to elicit NF-κB activity on their own. CONCLUSIONS: Skin barrier disruption activates the IL-1 and the PAR-2 pathways, which act in concert to activate the TSLP promoter and possibly other inflammatory genes. Awareness of this combined activity may permit a more flexible clinical management by selective targeting of either pathway individually or collectively. What's already known about this topic? Thymic stromal lymphopoietin (TSLP) is rapidly induced upon skin perturbation and mediates proallergic T helper 2-type responses by acting on leucocytes. Endogenous control of TSLP expression is poorly understood, but interleukin (IL)-1 is one regulator in the cutaneous environment In addition to IL-1, protease-activated receptor 2 (PAR-2) organizes central inflammatory pathways in the skin. What does this study add? IL-1 and PAR-2 pathways cooperate in driving TSLP production in mice and humans. Pathway integration occurs at the level of the TSLP promoter through enhanced recruitment and transcriptional activation of nuclear factor kappa B. When PAR-2 is co-stimulated, very low IL-1 levels (inactive by themselves) can induce biologically meaningful responses in the skin environment. What is the translational message? Physical skin irritation results in robust TSLP production by simultaneous activation of PAR-2 and IL-1 pathways.

Allergic FcεRI- and pseudo-allergic MRGPRX2-triggered mast cell activation routes are independent and inversely regulated by SCF.

While allergic mast cell (MC) degranulation occurs by FcεRI aggregation and varies in strength among subjects, the analogous pseudo-allergic route was recently uncovered to proceed via MRGPRX2. Here, we examine interindividual variability in skin MC responses to FcεRI triggering vs those evoked by MRGPRX2. While population-based variability is comparable between the routes, FcεRI- and MRGPRX2-stimulated pathways are completely independent from each other, and responsiveness to one has therefore no predictive value for the other. Conversely, degranulation triggered by compound 48/80 is highly correlated to the process elicited by substance P. MRGPRX2 mRNA shows pronounced population-based variability (coefficient of variation 102.9%). Surprisingly, stem cell factor (SCF) as the MC-supportive mediator par excellence potently inhibits pseudo-allergic degranulation, while it simultaneously promotes allergic stimulation via FcεRI. We conclude that SCF can have selective MC-dampening functions. Clinically, the data imply that subjects highly reactive in one pathway are not automatically hyper-responsive in terms of the alternative route.

Serum levels of 9α,11ß-PGF2 and apolipoprotein A1 achieve high predictive power as biomarkers of anaphylaxis.

Anaphylaxis is a life-threatening hypersensitivity reaction. To identify biomarkers for the condition, we assessed serum levels of apolipoprotein (Apo)A and ApoE. We found a reduction of both lipoproteins in anaphylactic mice as well as in orally challenged food allergic patients. We then compared patients after acute anaphylaxis with several control groups (nonallergic, history of allergen-triggered anaphylaxis, acute cardiovascular/febrile reactions). In this unpaired setting, ApoE levels were unaltered, while ApoA1 was reduced in the anaphylactic group. Although unable to discriminate between anaphylaxis and cardiovascular/febrile reactions, ROC curve analysis revealed a reasonably high area under the curve (AUC) of 0.91 for ApoA1. Serum 9α,11ß-PGF2 , recently identified as a suitable biomarker for anaphylaxis, outperformed ApoA1 with AUC=0.95. Intriguingly however its power further increased upon combination of both mediators reaching AUC=1. Our data suggest that ApoA1 combined with 9α,11ß-PGF2 represents a useful composite biomarker of anaphylaxis, achieving superior diagnostic power over either factor alone.

Thymic stromal lymphopoietin induction by skin irritation is independent of tumour necrosis factor-α, but supported by interleukin-1.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an extensively studied cytokine linked to the pathogenesis of allergic diseases, but the inherent activities behind TSLP expression are not well defined. OBJECTIVES: To explore the conditions favourable to TSLP induction outside of a typically allergic set-up and determine the associated mechanisms, and to assess whether TSLP is similarly controlled in murine and human skin. METHODS: A combination of primary keratinocytes, skin explants/epidermal sheets and in vivo strategies was employed. The skin of wild-type and tumour necrosis factor knockout (TNF-/-) mice was subjected to acute irritation. Cells and specimens were stimulated with a range of TSLP inducers in the presence or absence of neutralizing antibodies. TSLP was quantitated by quantitative reverse-transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: In addition to cytokines, skin irritation brought about by various causes (e.g. shaving, scratching and chemical perturbation) elicited uniformly high-level production of TSLP, which entered the circulatory system. Despite the potency of TNF-α as an in vitro TSLP inducer, the use of TNF-/- mice revealed that this mechanism was completely independent of endogenous TNF-α. Conversely, irritation-elicited TSLP depended on interleukin (IL)-1, which had a more pronounced influence in human skin than in murine skin. Murine and human skin differed considerably regarding TSLP regulation. CONCLUSIONS: Thymic stromal lymphopoietin is a general responder to disrupted skin homeostasis and may have a role in triggering the alarm system of the skin. TSLP induction is rapid, transient and driven by a mechanism that does not involve TNF-α, but partially relies on the evolutionarily ancient IL-1 system. The irritated skin secretes TSLP into the circulatory system. TSLP regulation varies between species.

Targeting the vitamin D receptor inhibits the B cell-dependent allergic immune response.

BACKGROUND: 1α,25-dihydroxyvitamin D(3) (calcitriol), the biologically active form of vitamin D, is an immunomodulatory hormone, e.g. it inhibits IgE synthesis in B cells. As its clinical application is limited by hypercalcemia, synthetic vitamin D receptor (VDR) agonists that mediate immunomodulatory activities without adverse hypercalcemic effects are of great interest. This study aimed to investigate the impact of a low-calcemic VDR agonist on the IgE immune response in vitro and in vivo. METHODS: Human peripheral B cells were cultured under IgE inducing conditions in the presence of VDR ligands. B cells were analyzed by quantitative RT-PCR, enzyme-linked immunosorbent assays, enzyme-linked immunospot technique, and flow cytometry. BALB/c mice were sensitized with ovalbumin (OVA)/alum followed by the therapeutic VDR agonist treatment and analyzed regarding the humoral immunoglobulin profile. RESULTS: The natural VDR ligand calcitriol, but also a low-calcemic VDR agonist, profoundly suppressed IgE production by human peripheral B cells by 63.9 ± 5.9%. The potential mechanisms involved are the reduction of the transcript for activation-induced deaminase (AID) and the reduction of IgE immunoglobulin-secreting cells by 68.1 ± 12.7%. By using an in vivo approach, we finally demonstrate that the humoral IgE response in a type I allergy mouse model was impaired by the VDR agonist. CONCLUSION: Our results show that targeting the VDR modulates the humoral immune response including IgE. Whether it might be useful for clinical applications has to be determined in appropriate clinical trials.

Tamoxifen counteracts the allergic immune response and improves allergen-induced dermatitis in mice.

BACKGROUND: Tamoxifen (TX) represents the prototype selective oestrogen receptor modulator. In addition to its use in breast cancer, TX possesses immunomodulatory functions and displays beneficial effects in models of systemic lupus erythematosus. We hypothesized that TX might inhibit type I allergic reactions, which are also characterized by deviations in humoral immunity. OBJECTIVE: To evaluate the effects of TX on the allergic immune response in appropriate mouse models. METHODS: Balb/c mice were sensitized with ovalbumin (OVA)-alum by the intraperitoneal route, and humoral parameters, T cell cytokine patterns and OVA-induced ear swelling responses were determined in a preventive (start of TX treatment before sensitization) and a therapeutic setting (start after sensitization), respectively. In addition, the impact of TX on clinical signs, epidermal thickness and leucocyte infiltration of the skin was investigated in a model of allergen-induced dermatitis. RESULTS: Preventive TX treatment interfered with all aspects of the allergic immune response, leading to a reduction of allergen-specific Ig levels (IgE, IgG1 and IgG2a), a skewing effect in the T cell compartment with the inhibition of IL-4 and an abrogation of ear swelling responses. Interestingly, a therapeutic TX administration was also effective in reducing Ig levels and ear swelling responses. The vigorous systemic effects were additionally mirrored by local changes in allergen-dependent dermatitis with reduced clinical symptoms, diminished epidermal thickness and decreased CD4+ and CD8+ cell infiltrates. CONCLUSION: TX inhibits allergic responses when given preventively and also therapeutically, and improves allergen-induced dermatitis. Because of its effectiveness, TX could bear significant therapeutic potential for the treatment of allergies.

The transcription factor profile of human mast cells in comparison with monocytes and granulocytes.

Expression profiles of mRNAs and proteins for various transcription factors were determined for human skin mast cells (sMCs), leukemic HMC-1 MCs, monocytes and granulocytes. By quantitative RT-PCR, sMCs expressed lower levels of c-fos, PU.1, C/EBPalpha, and C/EBPepsilon than monocytes and granulocytes, but higher levels of MITF, SCL, GATA-1 and GATA-2. At the protein level, MITF, SCL, GATA-2, Elf-1 and c-fos were clearly detectable in sMCs. With the exception of c-fos, these proteins were absent or expressed only slightly in monocytes and granulocytes. The expression of NF-E2p45, GATA-1, PU.1, Ets-1, C/EBPalpha and C/EBPepsilon was below the detection limit in sMCs, but detectable in other myelocytes. The high expression of SCL and GATA-2 in sMCs is reminiscent of stem cells. The absence of C/EBPvarepsilon in sMCs, but strong expression in HMC-1, suggests it may impair MC maturation. In summary, mature human MCs can be characterized as C/EBPalpha(low), C/EBPvarepsilon-, PU.1(low), GATA-1(low), GATA-2+, SCL+, MITF(high).

[Dynamics of some characteristics of humoral immunity, vaginal interferon status in pregnant women with the relapse of chronic pyelonephritis]. / Nekotorye pokazateli gumoral'nogo immuniteta, interferonovogo statusa vlagalishcha v dinamike u beremennykh s retsidivom khronicheskogo pielonefrita.

65 pregnant women with the exacerbation of chronic pyelonephritis in the III trimester of gestation and 34 healthy pregnant women were examined. The quantitative content of immunoglobulins, the activity of interferon in cervico = vaginal washings and the composition of the vaginal microflora were determined. All patients with the relapse of chronic pyelonephritis exhibited disturbances in the normal microbiocenosis of the genitals and the dysfunction of the local immunity of the genital system, accompanied with a decrease in serum and secretory IgA, an increase in the amount of IgG and IgM, increased interferon activity. Pregnant women with the relapse of chronic pyelonephritis received, in addition to traditional therapy, local treatment with Kipferon suppositories, an immunomodulating preparation. The study revealed that the use of this preparation normalized the characteristics of local immunity, the composition of the microflora' of the genitals and led to the disappearance of the clinical symptoms of the disease.

Retinoic acid up-regulates myeloid ICAM-3 expression and function in a cell-specific fashion--evidence for retinoid signaling pathways in the mast cell lineage.

Investigation of mast cell responsiveness toward retinoic acid (RA) revealed selective promotion of ICAM-3 expression in the human mast cell line HMC-1. This process was dose- and time-dependent and detectable by flow cytometry, Western blot analysis, ELISA, and Northern blot analysis. ICAM-3 modulation was found to be cell-type dependent, detectable also for HL-60 cells and monocytes but not U-937 and only weakly for KU812 cells. Terminally differentiated skin mast cells also failed to up-modulate their ICAM-3, suggesting the requirement for some degree of immaturity for the process. RA-mediated effects on ICAM-1 expression, studied in parallel, were clearly distinct from those on ICAM-3. Investigation of retinoid receptor expression, known to mediate intracellular RA signaling, revealed presence of RAR alpha, RAR gamma, RXR beta, and RXR gamma transcripts in all cell lines studied, and HMC-1 cells were the only line lacking RXR alpha. RAR beta, not expressed at baseline, was induced by RA in a fashion obviously correlating with ICAM-3 up-regulation. Increased ICAM-3 expression was of functional significance, such that processes stimulated or co-stimulated via ICAM-3 (homotypic aggregation, IL-8 secretion) were clearly enhanced upon RA pretreatment, suggesting that RA may contribute via hitherto unrecognized pathways to immune function and host defense.

Mast cells as initiators of immunity and host defense.

Until recently, mast cells have been viewed primarily as harmful because of their key role as effector cells of allergic and potentially lethal anaphylactic reactions. Their contribution to human health appeared instead to be limited to the elimination of parasites. There is, however, growing evidence for additional beneficial functions of mast cells, particularly regarding the initiation of acquired immune reactions. Thus, mast cells can phagocytize diverse particles, take up antigens, and express a number of receptors, particularly MHC class I and II antigens, ICAM-1 and -3, CD43, CD80, CD86 and CD40L which allow them to interact with T and B lymphocytes. They can also secrete numerous cytokines that induce and enhance recruitment and functions of lymphocytes. Finally, there is good evidence that mast cells present e.g. pollen and bacterial antigens, respond to bacterial superantigens, but fail to react to endogenously produced antigens or superantigens. Mast cells can also activate B cells directly to produce IgE, but this activity and the ability to produce IL-4 or IL-13 is restricted primarily to basophil leukocytes and mucosal mast cells. Finally, recent evidence attributes a pivotal role to the cells in natural immunity to bacteria. There is also emerging evidence that mast cells can downmodulate the immune response. While these data require further clarification, the basic ability of mast cells to initiate innate and acquired immune reactions can no longer be questioned.

Human leukemic (HMC-1) mast cells are responsive to 1alpha, 25-dihydroxyvitamin D(3): selective promotion of ICAM-3 expression and constitutive presence of vitamin D(3) receptor.

Expression levels of adhesion molecules on HMC-1 mast cells were examined prior to and following administration of 1alpha, 25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. While most receptors (including ICAM-1) remained unchanged by the treatment, solely ICAM-3 expression was promoted in a dose- and time-dependent fashion, peaking at 50 nM of 1,25(OH)(2)D(3) and 72 h, illustrating that like other myeloid cells, human mast cells are 1,25(OH)(2)D(3) responsive, yet in a highly selective manner. Flow cytometric results were confirmed by ELISA, by semiquantitative RT-PCR, and functionally by showing enhanced anti-ICAM-3 mediated homotypic aggregation of 1,25(OH)(2)D(3) pretreated cells. Since cellular responsiveness is conferred by the vitamin D(3) receptor (VDR), we examined human mast cells for its expression. VDR was constitutively present in both HMC-1 and skin mast cells by RT-PCR technique and in nuclear extracts of HMC-1 cells by Western blot analysis. Our data thus suggest that human mast cells are direct targets of 1, 25(OH)(2)D(3) action.

ICAM-3 (CD50) is expressed by human mast cells: induction of homotypic mast cell aggregation via ICAM-3.

Intercellular adhesion molecule-3 (ICAM-3, CD50), an adhesion receptor of the immunoglobulin superfamily, is suggested to play a key role in adhesive cellular interactions during the initial phase of an immune response. We here provide evidence that ICAM-3 is abundantly expressed by cells of the human mast cell line HMC-1 and, to a lower degree, by purified skin mast cells, as demonstrated by flow-cytometry, ELISA and RT-PCR. ICAM-3 immunoprecipitated from surface biotinylated HMC-1 cells migrates as a broad band of Mr 124,000 by Western blot analysis. We also demonstrate that monoclonal antibodies directed against ICAM-3 are capable of inducing rapid HMC-1 cell aggregation, the extent of which strongly depends on the epitope recognized by the mAb applied. Interestingly, although inhibitable by two of six mAbs against LFA-1, HMC-1 aggregation induced via ICAM-3 appears to be mediated by an adhesive pathway independent of LFA-1. Dermal mast cells are also aggregated with anti-ICAM-3 mAbs, a phenomenon which has not been described before for isolated tissue mast cells. However, this process displays slower kinetics, as compared to HMC-1 cells. That anti-ICAM-3 mAbs are able to mediate biological effects is further illustrated by their capability to increase stimulation-dependent release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 from HMC-1 cells. Taken together, these results indicate that ICAM-3 is not only expressed by immature and mature human mast cells, but also possesses functional relevance and may therefore play a significant role in mast cell associated processes.

Signal transduction via CD43 (leukosialin, sialophorin) and associated biological effects in human mast cell line (HMC-1).

CD43 has been shown to be involved in the regulation of cellular adhesion and activation of leukocytes, but its functional significance for mast cell biology has been poorly defined. We demonstrate here that mAb engagement of surface CD43 on human leukemic (HMC-1) mast cells initiates a signaling cascade which involves protein kinase C, while tyrosine kinases appear to play a minor role, as evidenced by effects of different kinase inhibitors on homotypic aggregation induced via CD43. Furthermore, administration of an activating anti-CD43 mAb is shown to induce and promote TNF-alpha- and to enhance IL-8-secretion from HMC-1 cells, but it does not initiate histamine, tryptase, or LTC4 release, suggesting that the intracellular pathways leading to aggregation and release of certain mast cell mediators are differentially regulated. Additionally, engagement of CD43 on HMC-1 cells leads to down-regulation of CD43 surface expression, implying that CD43 may be potentially involved in its own regulation.

Leukosialin (CD43) is proteolytically cleaved from stimulated HMC-1 cells.

Leukosialin (CD43), the major sialoprotein on circulating leukocytes, has been previously described to be down-regulated on neutrophils following activation with phorbol myristate acetate (PMA). The other single cells previously examined, blood lymphocytes, do not down-regulate CD43 when stimulated by PMA. Recently, we have characterized leukosialin on the human mast cell line HMC-1 and observed that leukosialin is down-regulated after stimulation with PMA. In the present study, we have investigated the mechanism of PMA-mediated down-regulation of CD43 on HMC-1 cells (subclone 5C6). PMA caused the release of soluble leukosialin (123 kD) during HMC-1 cell activation. The molecular weight of soluble leukosialin was nearly identical to that of the cell-membrane bound molecule, suggesting a cleavage proximal from the cell membrane. Inhibitors of serine proteases, like phenylmethylsulphonyl fluoride (PMSF), benzamidine and 3, 4-dichloroisocoumarin, blocked the PMA-mediated cleavage of CD43. In all experiments, the inhibition of CD43-down-regulation was dependent on the concentration of protease inhibitors. Treatment of HMC-1 cells with various proteases (trypsin, alpha-chymotrypsin, elastase, papain, nagarse) substantially decreased anti-CD43 binding capacity and caused the release of soluble leukosialin (116 kD) or its fragments into the supernatant. Pretreatment of HMC-1 cells with neuraminidases from Vibrio cholerae or Arthrobacter ureafaciens resulted in an increased sensitivity of CD43 against proteases, whereas the effects of PMA were not influenced. In conclusion, proteolytic cleavage of CD43 is described for the first time in a cell other than neutrophils, namely HMC-1 cells. Our results suggest that serine proteases are involved in the PMA-mediated down-regulation of leukosialin on HMC-1 cells.

Human leukaemic (HMC-1) and normal skin mast cells express beta 2-integrins: characterization of beta 2-integrins and ICAM-1 on HMC-1 cells.

Mast cells are bone marrow-derived, ubiquitous connective tissue resident cells. However, their mechanisms of migration, the distribution of immature and mature cells and their interaction with other inflammatory cells are largely unclarified. Possibly, beta 2-integrins play an important role in these processes. In the present investigation, the authors studied the expression and regulation of the beta 2-integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18) and of the LFA-1/Mac-1 counter-receptor intercellular adhesion molecule-1 (ICAM-1; CD54) on leukaemic (HMC-1 cell subclone 5C6) and on normal mature human skin mast cells. The HMC-1 cells clearly expressed CD11a, CD18 and CD54, while expression of CD11b and CD11c was low. The apparent molecular weights were 180 kDa (CD11a), 95 kDa (CD18) and 90 kDa (CD54) as determined by Western blot analysis. Phorbol myristate acetate (PMA) induced a time- and dose-dependent up-regulation of CD11a, CD11b, CD11c, CD18 and CD54 that was inhibited by cycloheximide, suggesting a dependence on de novo protein synthesis. Enhanced expression of CD11a, CD11b, CD11c and CD18 could also be confirmed at the gene level as demonstrated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Increased expression of LFA-1/ICAM-1 in response to PMA was accompanied by strong enhancement of homotypic cell aggregation, suggesting that newly synthesized LFA-1/ICAM-1 is functionally active. In order to determine a physiologically relevant way of mast cell beta 2-integrin modulation, several cytokines and chemotactic mediators (interleukin-4, IL-4; nerve growth factor beta, NGF beta; C5a; and leukotriene B4, LTB4) were tested for their influence on adhesion molecule cell surface density. Only LTB4 was shown specifically to up-regulate CD11a and CD18, but not CD11b or CD11c. The presence of CD11a, CD11c and CD18 could be confirmed on a low percentage of normal skin mast cells by immunofluorescence, using a double staining technique. In comparison to normal skin, a significantly higher percentage of CD18+ mast cells was found in inflammatory dermatoses such as psoriasis vulgaris, atopic dermatitis and lichen planus. Therefore, mast cell beta 2-integrins possibly play an important role during homing of immature mast cells as well as during the interaction of activated mast cells with other inflammatory cells.

CD43 (leukosialin, sialophorin) expression is differentially regulated by retinoic acids.

CD43 (leukosialin, sialophorin), a cell-surface associated mucin that is constitutively expressed at high levels on most leukocytes, is thought to be involved in cell activation and adhesion. We here provide evidence that the vitamin A metabolites all-trans and 13-cis retinoic acid up-regulate CD43 on human leukemic (HMC-1) mast cells, as determined by flow cytometry, Western blot analysis, and by semiquantitative reverse transcriptase-polymerase chain reaction. Enhanced CD43 expression was accompanied by a strong increase in anti-CD43-mediated, LFA-1-dependent homotypic aggregation of HMC-1 cells, demonstrating that newly synthesized CD43 is functionally active in transmitting signals across the plasma membrane which result in enhanced cellular adhesion. CD43 expression was also enhanced in response to retinoic acids on isolated human skin mast cells and human monocytes, but not on cells of the basophilic cell line KU-812 and promyelocytic HL-60 cells, indicating that these agents might act in a cell-type specific manner. These combined result-point to a novel aspect in the regulation of CD43. Possibly, vitamin A metabolites act directly on the CD43 gene, since putative retinoic acid response elements have been detected within its regulatory regions.