The high-affinity IgE receptor (FcepsilonRI) blocks apoptosis in normal human monocytes.

Monocytes have a limited life span, and their homeostasis is regulated by apoptosis in vivo. When cultured in the absence of appropriate exogenous stimuli, they undergo apoptosis, but under the influence of survival signals, these cells differentiate into macrophages or dendritic cells. Here we show that ligation of the high-affinity IgE receptor (FcepsilonRI) on human monocytes from nonatopic individuals markedly reduces apoptosis induced by serum deprivation or by CD95/Fas ligation. Aggregation of FcepsilonRI reduces its own expression but fails to modulate CD95/Fas expression. In contrast, FcepsilonRI ligation enhances the expression of the antiapoptotic molecules Bcl-2 and Bcl-xL, but not Mcl-1, in monocytes. Incubation of unstimulated cells with culture supernatants of FcepsilonRI-activated monocytes prolongs their life span, whereas CD95/Fas expression remains unaffected. The incidence of apoptosis is restored considerably when the supernatant is depleted of TNF-alpha, whereas elimination of IL-1beta, GM-CSF, or IL-12 has no effect. These results indicate that FcepsilonRI mediates signals preventing monocyte apoptosis directly by increasing the levels of Bcl-2 and Bcl-xL, and indirectly by means of TNF-alpha in an autocrine and paracrine fashion. This process may contribute to the establishment of chronic allergic disorders such as atopic dermatitis.

The human diploid fibroblast senescence pathway is independent of interleukin-1 alpha mRNA levels and tyrosine phosphorylation of FGFR-1 substrates.

In vitro cellular senescence of human umbilical vein endothelial cells (HUVEC) may involve the intracellular activity of the signal peptide-less cytokine interleukin (IL)-1 alpha. To determine whether senescence of other human diploid cells involves the function of IL-1 alpha, we examined the steady-state expression of IL-1 alpha mRNA in IMR-90 fibroblasts. The IL-1 alpha transcript was not elevated in senescent IMR-90 cells. With the exception of the plasminogen activator inhibitor (PAI)-1 transcript, other IL-1 alpha-response gene mRNAs were not induced in senescent IMR-90, although the mRNA for each gene was induced by exogenous IL-1 alpha. The mRNA expression of cell cycle-specific genes demonstrated that Fos and ornithine decarboxylase (ODC) were induced in young and senescent cells in response to both serum and fibroblast growth factor (FGF)-1. Histone (H)3 mRNA was induced by serum in young cells, but not in senescent cells, and FGF-1 failed to induce H3 mRNA in either young or senescent cells. Further, while young IMR-90 populations were able to respond to serum as an initiator of DNA synthesis and cell growth, they did not exhibit a response to exogenous FGF-1. FGF receptor (R)-1 substrates were not tyrosine phosphorylated in either young or senescent IMR-90 cells. These data demonstrate that IL-1 alpha and FGF-1 may have different functions in HUVEC and IMR-90 fibroblast populations including distinct pathways for the regulation of cellular growth and senescence.

Regulation of the high affinity receptor for IgE on human epidermal Langerhans cells.

Human epidermal Langerhans cells (LC) express variable amounts of the high affinity receptor for IgE (Fc epsilonRI); the strongest expression is characteristic of atopic dermatitis. The receptor is suggested to take part in the pathophysiology of this disease by acting as a link between aeroallergens and Ag-specific T cells in an IgE-mediated, delayed-type hypersensitivity reaction. In the present study we show that even in the absence of surface expression, normal LC maintain an intracellular pool of the alpha-chain of Fc epsilonRI (Fc epsilonRIalpha) of the same m.w. as the surface-bound Fc epsilonRIalpha that is able to bind significant amounts of IgE. The lack of surface expression is linked to the absence or very low expression of the gamma-chain (Fc epsilonRIgamma). Moreover, the amount of Fc epsilonRIalpha expressed at the cell surface significantly correlates with the amount of Fc epsilonRIgamma. LC differentiation toward lymphoid dendritic cells is accompanied by the disappearance of transcripts for Fc epsilonRIalpha, but not for Fc epsilonRIgamma. This leads to a rapid decrease in the intracellular and surface levels of Fc epsilonRIalpha, which cannot be influenced by IL-4, IgE, or other agents. Overall, our findings suggest that these mechanisms enable LC to be highly versatile APCs by rapidly adapting the surface level of Fc epsilonRI to distinct inflammatory environments.

Post-transcriptional regulation of interleukin 1 alpha in various strains of young and senescent human umbilical vein endothelial cells.

Human umbilical vein endothelial cell (HUVEC) senescence in vitro is characterized by the loss of proliferative potential and an increase in cell size. Because HUVEC senescence in one strain (H101) has been characterized by the increase in the steady-state mRNA level for the signal-peptideless cytokine, interleukin (IL) 1 alpha, we have examined young and senescent populations of five additional HUVEC strains (H3605, H103, H928, H929, and H930) to determine whether the elevated levels of IL-1 alpha mRNA could be observed in all HUVEC strains. Consistent with the data from strain H101, strains H3605 and H930 also exhibited a low steady-state level of the IL-1 alpha mRNA in young populations compared to elevated levels of IL-1 alpha mRNA in the senescent populations. However, three strains (H103, H928, and H929) did not exhibit reduced levels of IL-1 alpha mRNA in the young populations, and interestingly, strain H928, at times, expressed relatively high IL-1 alpha mRNA levels in the young populations. In addition, expression of the steady-state level of plasminogen activator inhibitor 1 and cyclooxygenase 2 was elevated in senescent populations of all HUVEC strains examined, whereas young populations exhibited a low level of expression for these genes regardless of the IL-1 alpha mRNA level. Further, the level of the IL-1 alpha polypeptide was elevated in senescent HUVEC populations relative to young populations that expressed either a high or low level of the IL-1 alpha mRNA. We have also demonstrated that the elevated level of IL-1 alpha mRNA in the senescent population of strain H3605 may be regulated by mRNA stability; however, this mechanism does not apply to all the HUVEC strains examined in this study. Thus, we suggest that while mRNA levels of the IL-1-response genes for plasminogen activator inhibitor 1 and cyclooxygenase 2 are appropriate markers for HUVEC senescence, HUVEC strain-specific post-transcriptional mechanisms may exist to regulate the function of IL-1 alpha as a modifier of HUVEC senescence in vitro.

Identification of a nuclear localization sequence within the structure of the human interleukin-1 alpha precursor.

Recent studies have suggested that the signal peptide-less cytokine, interleukin (IL)-1 alpha, may play a role as an intracellular regulator of human endothelial cell proliferation in vitro (Garfinkel, S., Haines, D. S., Brown, S., Wessendorf, J., Gillespie, D. H., and Maciag, T. (1992) J. Biol. Chem. 267, 24375-24378). In order to determine the intracellular locale of the IL-1 alpha precursor, we fused the open reading frame of the IL-1 alpha precursor to the reporter gene beta-galactosidase (Gal) and studied the cellular distribution of the chimera in NIH 3T3 cells after transfection. Immunological and enzymatic analysis demonstrated that the IL-1 alpha:beta-Gal fusion protein was associated with the nucleus. To further define the region responsible for this activity, we ligated the mature form of IL-1 alpha (IL-1 alpha 113-271) and the IL-1 alpha precursor domain (IL-1 alpha 1-112) to beta-Gal. Analysis of the intracellular distribution of these chimeric polypeptides following transfection demonstrated a differential distribution of IL-1 alpha 1-112:beta-Gal in the nucleus and IL-1 alpha 113-271:beta-Gal in the cytosol. Because the IL-1 alpha precursor domain contains a sequence that resembles a nuclear translocation signal (KVLKKRRL, residues 79-86), we prepared an IL-1 alpha precursor point mutant in which Lys82 was replaced by Glu. Transfection of NIH 3T3 cells with the IL-1 alpha precursor point mutant (IL-1 alpha 1-271 Glu82:beta-Gal) resulted in a significant reduction in the ability of the IL-1 alpha precursor to associate with the nucleus and similar data were obtained as a result of Lys82 mutagenesis in the IL-1 alpha precursor domain (IL-1 alpha 1-112 Glu82:beta-Gal). These data suggest that the IL-1 alpha precursor contains a functional nuclear localization sequence within the structure of the precursor domain and Lys82 is critical for its function.

IL-4 induces the intracellular expression of the alpha chain of the high-affinity receptor for IgE in in vitro-generated dendritic cells.

BACKGROUND: Recent findings have shown that the surface expression of the high-affinity receptor for IgE (FcepsilonRI) on human CD1a(+) Langerhans cells (LC) and related dendritic cells (DC) in the skin, despite a constant intracellular expression of its alpha chain (FcepsilonRIalpha), is highly up-regulated in atopic dermatitis. Moreover, this surface expression correlates with the IgE serum level, strongly suggesting yet-to-be-defined common signals in the regulation of FcepsilonRI display on LC/DC and IgE synthesis. OBJECTIVES: In this study we examined the influence of different cytokines on the expression of FcepsilonRI on in vitro-generated CD1a(+) LC/DC. METHODS: CD34(+) precursor cells were isolated from cord blood with use of high-gradient magnetic cell sorting, cultured with GM-CSF, TNF-alpha, IL-4, or IFN-gamma, and surface and cytoplasmic staining for flow cytometry were performed. RESULTS: IL-4 strongly enhanced the generation of CD1a(+) LC/DC and also up-regulated the expression of the skin-homing structures E-cadherin and cutaneous lymphocyte antigen. In contrast, IFN-gamma was found to suppress the E-cadherin expression and to be a strong antagonist of IL-4 by inhibiting the production of CD1a(+) cells. Most important, IL-4 induced the cytoplasmic expression of FcepsilonRIalpha in CD1a(+) LC/DC but not its surface expression. This up-regulation was antagonized by IFN-gamma. CONCLUSION: IL-4 is not only a key cytokine in the regulation of IgE but also induces the expression of its receptor binding chain as well as up-regulation of skin homing molecules on LC/DC. Expression of these structures during generation of LC/DC reflects the in vivo situation encountered in LC.