Diverse gram-negative bacterial cells communicate with each other by using diffusible N-acyl homoserine lactone (AHL) signal molecules to coordinate gene expression with cell population density. Accumulation of AHLs above a threshold concentration renders the population “quorate,” and the appropriate target gene is activated. In pathogenic bacteria, such as Pseudomonas aeruginosa, AHL-mediated quorum sensing is involved in the regulation of multiple virulence determinants. We therefore sought to determine whether the immune system is capable of responding to these bacterial signal molecules. Consequently the immunomodulatory properties of the AHLsN-(3-oxododecanoyl)-l-homoserine lactone (OdDHL) and N-(3-oxohexanoyl)-l-homoserine lactone (OHHL) were evaluated in murine and human leukocyte immunoassays in vitro. OdDHL, but not OHHL, inhibited lymphocyte proliferation and tumor necrosis factor alpha production by lipopolysaccharide-stimulated macrophages. Furthermore, OdDHL simultaneously and potently down-regulated the production of IL-12, a Th-1-supportive cytokine. At high concentrations (>7 × 10−5 M) OdDHL inhibited antibody production by keyhole limpet hemocyanin-stimulated spleen cells, but at lower concentrations (<7 × 10−5 M), antibody production was stimulated, apparently by increasing the proportion of the immunoglobulin G1 (IgG1) isotype. OdDHL also promoted IgE production by interleukin-4-stimulated human peripheral blood mononuclear cells. These data indicate that OdDHL may influence the Th-1–Th-2 balance in the infected host and suggest that, in addition to regulating the expression of virulence determinants, OdDHL may contribute to the pathogenesis of P. aeruginosa infections by functioning as a virulence determinant per se.
Soluble CD23 (sCD23) has been proposed to play an important role in the up-regulation of immunoglobulin E (IgE) synthesis. Production of sCD23 is dependent on the proteolytic cleavage of membrane CD23, but the protease(s) involved in this process remain unknown. Preliminary data, obtained by testing a panel of protease inhibitors, suggested that this enzyme may be a zinc-dependent metalloproteinase. Therefore, we investigated the effect of a standard hydroxamate-type Zn2+ metalloproteinase inhibitor (GI 129471) on both sCD23 and IgE release from human tonsillar B cells, stimulated with interleukin-4 (IL-4) and anti-CD40. Incubation of cells for 3 days with GI 129471 inhibited the production of sCD23 with an IC50 of 602 nm+/-3 nm (n=3), but by 14 days the activity of the compound against sCD23 had decreased by greater than threefold (IC50 2+/-0.26 microM; n=3). On the other hand, GI 129471 caused a potent inhibition of IgE production, with no apparent loss of activity over the culture period (14 days: IC50 250 nm+/-72 nm; n=3). Time-course studies showed that, despite loss of activity against sCD23, inhibition of sCD23 production early in the culture was able to cause a potent and long-lasting inhibitory effect on IgE. Furthermore, we also showed that the activity of GI 129471 is selective for IgE, as no effect was seen on immunoglobulin G1 (IgG1) or IgG4 production at test concentrations as high as 10 microM. These results support the hypothesis that metalloproteinases may be involved in the proteolytic cleavage of CD23 and subsequent regulation of IgE synthesis. Inhibition of the protease(s) responsible for such cleavage may be of value in the treatment of allergic disease.
The development of these systems provides directly comparable T cell-containing and T cell-depleted cultures for human IgE synthesis from peripheral blood, allowing further study of the role of T cells in IgE regulation. These systems will also be of use for determining whether potential modulators of IgE synthesis act on the T cells or on other cell types.
SUMMARYIn this study, we investigated the moduiatory effects of CsA on in vitro synthesis of IgE. IgGI and lgG4 by human peripheral blood mononuclear cells (PBMC). In contrast to its known immunosuppressive elTect, we have demonstrated that a low dose of CsA (IO'^M. l20ng/ml) potentiated IgE production by up to 40-fold (i.e. from 33±4 5 to 1346±290 ng/nil). This potentiation was specific for IgE, since no such effect was demonstrable with IgGI and IgG4. Potentiation of IgE synthesis by CsA in the PBMC cultures was partly due to CsA acting on T cells, as demonstrated by the addition of CsA-treated T cells to T cell-depleted cultures. However, potentiation was also demonstrable in a T cell-depleted. anti-CD40-stimulated culture (four-fold increase from 400 ± 48 to 1606 ± 127 ng/nil). Our data therefore suggest that there are at least two mechanisms for CsAinduced potentiation of IgE synthesis, one T cell-dependent and the other T cell-independent. The clinical implications of these findings are discussed with regard to the use of CsA in the treatment of Th2-mcdiated diseases.
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