The 'monocytic' cell line I937 displays typical B cell characteristics.

The monocytic cell line I937 was derived from U937 by sorting for cells with high expression of MHC class II molecules. Further analysis of these class II molecules revealed the presence of the HLA-DR3 subtype suggesting that the cell line was a potential candidate for testing antigen presentation to T cells restricted by HLA-DR3. We found that the T cell clones CFTS4:2.80 and CFTS4:2.6 with the required restriction element responded to the house dust mite antigen DPT presented by I937 but not U937, whereas CFTS4:3.1, which is not HLA-DR restricted, did not respond to either cell line. Subsequent analysis of surface markers on I937, however, indicated that the cell line is of B cell origin. In contrast to the parental cell line U937, I937 was tested negative for CD4, CD31 and CD64 but expressed CD19, CD21 and CD40. Although neither surface nor cytoplasmic Ig molecules were detected in either I937 or U937, Southern blot analysis revealed IgH gene rearrangement in I937. In addition, a fragment specific for Epstein-Barr virus nuclear antigen (EBNA2) was amplified in I937 by PCR technique. Therefore, we conclude that I937 is an EBV-transformed B cell line, presumably derived from the same donor and not as reported originally as a subline of U937, which expresses high MHC class II levels.

Molecular consequences of human mast cell activation following immunoglobulin E-high-affinity immunoglobulin E receptor (IgE-FcepsilonRI) interaction.

The cross-linking by immunoglobulin E of its high-affinity receptor, FcepsilonRI, on mast cells initiates a complex series of biochemical events leading to degranulation and the synthesis and secretion of eicosanoids and cytokines through the action of transcription factors, such as nuclear factor-kappaB. The initial activation involves the phosphorylation of FcepsilonRI beta- and gamma-subunits through the actions of the tyrosine kinases lyn and syk. For the purposes of description, the subsequent events may be grouped in three cascades characterized by the key proteins involved. First, the phospholipase C-inositol phosphate cascade activates protein kinase C and is largely responsible for calcium mobilization and influx. Second, activation of Ras and Raf via mitogen-activated protein kinase causes the production of arachidonic acid metabolites. Third, the generation of sphingosine and sphingosine-1-phosphate occurs through activation of sphingomyelinase. While the early signaling events tend to be specific for the cited cascades, there is an increasing overlap of activated proteins with the downstream propagation of the signal. It is the balanced interaction between these proteins that culminates in degranulation, synthesis, and release of eicosanoids and cytokines.

Modulation of Fc gamma receptor-mediated early events by the tyrosine phosphatase CD45 in primary human monocytes. Consequences for interleukin-6 production.

Stimulation of primary human monocytes from several donors by cross-linking of Fc gamma receptor type I (Fc gamma RI) and Fc gamma RII gave rise to calcium mobilization and protein tyrosine phosphorylation. These early events were not observed without cross-linking. CD45, a transmembrane tyrosine phosphatase, when co-cross-linked with either Fc gamma RI or Fc gamma RII, could prevent Fc gamma RI and Fc gamma RII-mediated calcium mobilization and protein tyrosine phosphorylation. When interleukin (IL)-6 production was measured, we noted a strong IL-6 production after activation of primary human monocytes by cross-linking of Fc gamma RI or Fc gamma RII. In contrast to calcium mobilization and tyrosine phosphorylation of proteins, IL-6 production was not affected by co-cross-linking of CD45 with either Fc gamma RI or Fc gamma RII. Interestingly, cross-linking of the CD45 itself was sufficient to induce IL-6 production. Our results show that the CD45 molecule is important in modulating early events following stimulation of primary human monocytes by cross-linking of Fc gamma RI or Fc gamma RII. However, triggering of CD45 alone can also induce IL-6 production, indicating that CD45 ligation itself can give signals and may have an important role in cytokine induction pathways.

Characterization of Fc epsilonRI expressing human monocytic cell lines. 1. The role of CD45 on signal transduction in primary monocytes and cell lines.

BACKGROUND: Recently, the high affinity receptor for IgE (Fc epsilonRI), which plays a major role in allergies, has been identified on a number of different antigen-presenting cell types, including human monocytes from atopic and nonatopic donors. In this report human monocytic cell lines were used to test for the expression of Fc epsilonRI, reasoning that a monocytic cell line expressing Fc epsilonRI constitutively would be a useful tool for large scale studies on the regulation of IgE binding and signal transduction. METHODS: Reverse transduction polymerase chain reaction was applied to identify Fc epsilonRI alpha-chain message, flow cytometry to detect Fc epsilonRI surface expression and signal transduction on the cell lines generated by transfection. RESULTS: We report the establishment of monocytic cell lines constitutively expressing Fc epsilonRI (THP1-alpha01 to THP1-alpha40) generated by transfection of the cell line THP1 with a plasmid encoding the Fc epsilonRI alpha-chain only. Fc epsilonRI on the THP1-alpha lines specifically binds IgE and is functional with regard to ligand binding and signal transduction. Comparative studies between the transfectants and primary human monocytes from nonatopic donors demonstrated the regulatory role of the tyrosine phosphatase CD45 on Fc epsilonRI-mediated cell activation. CONCLUSIONS: Monocytic cell lines carry Fc epsilonRI alpha-chain RNA and enhancement by transfection results in surface Fc epsilonRI expression on THP1. Triggering the receptor on the THP1-alpha lines or on human monocytes, which express native Fc epsilonRI, elicits a rapid and transient calcium mobilization, prevented by co-cross-linking of Fc epsilonRI and CD45.

Function and regulation of Fc epsilon RI expression on monocytes from non-atopic donors.

BACKGROUND: The high affinity receptor for IgE (Fc epsilon RI) has recently been identified on antigen presenting cells, i.e. Langerhans cells and monocytes from atopic donors and it was hypothesized that Fc epsilon RI expression levels correlated with allergy. OBJECTIVE: The aims of the study was to investigate the function and expression of Fc epsilon RI on monocytes from non-atopic donors. METHODS: Purified monocytes or peripheral blood mononuclear cells were used to study Fc epsilon RI expression and signal transduction on CD14 positive cells by flow cytometry and/or confocal laser microscopy. RESULTS: Freshly isolated monocytes from healthy individuals (n = 58) were shown to express Fc epsilon RI (median 18%, range 2-66%). No IgE was bound to these receptors in vivo, and in vitro no significant binding of complete IgE molecules could be obtained. IgE positive monocytes from atopic donors were also found to have free Fc epsilon RI incapable of binding IgE in vitro. On all CD14 positive cells free Fc epsilon RI expression was rapidly and completely lost during culture in conventional culture media (IMDM, RPMI) but not in phosphate buffered saline (PBS). Moreover, signal transduction through free Fc epsilon RI appeared to be inhibited. However, both IgE binding and calcium mobilization were restored by treatment of fresh non-atopic monocytes with neuraminidase. Importantly, culturing these monocytes overnight in conventional medium containing 2 micrograms/mL IgE induced a cycloheximide insensitive accumulation of IgE bound to Fc epsilon RI and, in addition, led to cell activation. CONCLUSION: Monocytes from both atopic donors and healthy individuals express Fc epsilon RI, but the previously reported different expression levels between the two groups seem to be directly related to the absence or presence of IgE in the serum. This may be due to the fact that Fc epsilon RI is subjected to a constant turnover process which is slowed down but not prevented by ligand binding. In addition, free Fc epsilon RI on non-atopic monocytes are under control of a neuramindase sensitive structure(s), which influences signal transduction and IgE binding.

Regulation of Fc epsilonRI expression on human monocytic cells by ligand and IL-4.

BACKGROUND: The Fc epsilonRI subunit composition and kinetic of expression differ between antigen-presenting cells and mast cells. Up to now, there has been no human in vitro model available that mimics the characteristics on monocytes. OBJECTIVE: The characterization of a natural human monocytic cell line (THP1), which expresses Fc epsilonRI, and the comparison to primary human monocytes and other monocytic cell lines, which only express Fc epsilonRI after transfection with the human Fc epsilonRI alpha-chain gene. METHODS: Surface receptor expression was characterized by flow cytometry, the human Fc epsilonRI alpha-chain gene was introduced by electroporation, and induction of Fc epsilonRI alpha-chain message was detected by semiquantitative RT PCR. RESULTS: Here we show that the parental human cell line THP1, but none of the other cell lines tested, displays surface Fc epsilonRI in response to IL-4 or incubation with receptor ligand (IgE, antibody). Transfection of Fc epsilonRI alpha-chain resulted in receptor expression on all cell lines, all of which increased surface Fc epsilonRI in the presence of IgE. Only the THP1-alpha transfectant, however, further increased receptor levels in response to IL-4, resulting from mRNA induction for the Fc epsilonRI-alpha, but not the beta- or gamma-subunit. CONCLUSION: Based on THP1, U937 and HL60 and their alpha-chain transfectants we present a model system for the study of Fc epsilonRI regulation and signalling on human cells. THP1 in particular, due to its responsiveness to both ligand and IL-4, even without prior manipulation, is ideally suited to address questions on Fc epsilonRI modulation in an 'allergic environment'.