Individuals with occupational allergy to detergent enzymes display a differential transcriptional regulation and cellular immune response.

BACKGROUND: In spite of significant safety measures, allergy to industrial enzymes remains a major concern. The increasing prevalence of occupational allergy emphasizes the need to investigate the functional properties of enzyme-exposed dendritic cells (DCs), as DCs possess a potent ability to activate allergen-specific T cells. OBJECTIVE: This study aims at elucidating the molecular mechanisms underlying allergic immune responses to lipase, an industrial enzyme. For this purpose, we studied the effect of both hypoallergenic and wild-type lipase on the transcriptional regulation in DCs and their stimulatory effect on memory CD4+ T cells. METHODS: Five individuals with documented lipase allergy were tested for specific serum IgE. DCs from these individuals, stimulated with lipases, were assayed for their ability to affect proliferation and polarization of memory T cells. The effect of lipases on transcriptional activity in DCs was evaluated using global expression analysis. RESULTS: Lipase-specific IgE levels varied considerably between donors, with donor 4 exhibiting highest levels, and a potent specific CD4+ T cell recall response was demonstrated only for donor 4. No difference was detected in cytokine profile when T cells from donor 4 were co-cultured with DCs pulsed with either hypoallergenic or wild-type lipase, as demonstrated by high IL-4 and IL-13, and low IFN-gamma production. However, the lipases induced different genetic signatures in DCs from donor 4, as compared with the non-responders. CONCLUSIONS: DCs from individuals with clinically diagnosed allergy to lipase displayed a differential response to stimulation with hypoallergenic and wild-type lipase in vitro. Only allergen-pulsed DCs from donor 4 were able to induce CD4+ T cell proliferation. The lipase-specific T cells displayed a T-helper type 2 phenotype, which was not altered by hypoallergenic lipase-pulsed DCs. Furthermore, DCs derived from donor 4 and stimulated with either of the lipases displayed different transcriptional profiles, as compared with the other donors. These signatures represent genes of potential importance for an immunoregulatory role of DC in an ongoing allergic response.

Bet v 1 homologues in strawberry identified as IgE-binding proteins and presumptive allergens.

BACKGROUND: No strawberry allergen has so far been identified and characterized. METHODS: Serum samples were collected from patients with a suggestive case history of adverse reactions to strawberry and other fruits. Extracts from fresh and frozen strawberries were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and mass spectrometry. Patient blood samples were analysed for inhibition of IgE binding and basophil degranulation. RESULTS: Several IgE-binding proteins could be detected. In more than half of the patient sera, a 20/18-kDa doublet band was observed in Western blotting. These two bands were excised and analysed by mass spectrometry showing the presence of proteins belonging to the Bet v 1 family of allergens. Inhibition of the IgE binding to the 20/18-kDa doublet was obtained by addition of two recombinantly expressed allergens belonging to the Bet v 1 family (Bet v 1 and Mal d 1) and strawberry protein extract. In a cell-based assay of patient blood samples, basophil degranulation could be induced by strawberry protein extract and by Bet v 1 and Mal d 1. CONCLUSIONS: We conclude that strawberry homologues to Bet v 1 may be allergens of importance for adverse reactions to strawberry.

Association of decreased phosphorylation of ERK-2 with costimulation of rat T cell activation by MEK-1 inhibitors and TGF-beta1.

Transforming growth factor-beta (TGF-beta) is usually known as an immunosuppressive cytokine, but we and others have shown stimulatory effects of TGF-beta on activation of Th2 T-lymphocytes. In the present investigation we have studied the effect of TGF-beta1 on phosphorylation of ERK, a MAP-kinase downstream of the Ras pathway. ERK is phosphorylated by MEK-1 and PD098059 and U0126 are specific inhibitors for this kinase. We demonstrate in the present study that these inhibitors abrogate the inhibitory effect of adh-splc (adherent-spleen cells) on activation of primary rat T-cells and induce a strong costimulatory effect almost as strong as we have previously shown with TGF-beta1. When TGF-beta1 is acting stimulatory on T-cell activation, it decreases phosphorylation of ERK-2 and thereby its activation. To investigate whether TGF-beta1 and MEK-1 inhibitors influence the same pathways, we compared their effects on cytokine profiles associated with SEA-induced rat T cell activation. TGF-beta1 induced IL-10 production, slightly decreased TNF-alpha production and decreased IFN-gamma production. The PD098059 inhibitor decreased both IFN-gamma and TNF-alpha production and together with TGF-beta1, it totally blocked IFN-gamma, TNF-alpha and IL-10 production. Thus TGF-beta1 and PD098059 showed overlapping but not identical effects on the cytokine pattern.

Effects of transforming growth factor beta1 expression in a rat colon carcinoma: growth inhibition, leukocyte infiltration and production of interleukin-10 and tumor necrosis factor alpha.

The cytokine transforming growth factor beta-1 (TGFbeta1), was transfected into a TGFbeta1-negative rat colon carcinoma. The growth of isografts of TGFbeta1-expressing tumors was compared to that of vector control transfectants. The TGFbeta1 transfectant grew significantly more slowly after intrahepatic isografting than did vector control and wild-type tumors. The TGFbeta1-transfected tumor tissue had significantly greater infiltration of both CD4+ and CD8+ T lymphocytes than did the vector control tumor. The tumor-infiltrating leukocytes (TIL) from TGFbeta1-transfected tumor secreted significantly more of the cytokines interleukin-10 (IL-10) and tumor necrosis factor alpha (TNFalpha) than did TIL from the vector control tumor. The TGFbeta1 transfectant also demonstrated a significantly slower outgrowth in immunodeficient SCID mice, supporting a non-T-lymphocyte-dependent mechanism for the tumor retardation. In SCID mice, the TGFbeta1-transfected tumor demonstrated significantly greater infiltration of both granulocytes and macrophages than did the vector control transfectant. We also demonstrated a direct inhibitory effect of rat TNFalpha on tumor proliferation in vitro. These results suggest that TGFbeta1 induces a local secretion of immunomodulating cytokines and that this may influence monocytes, lymphocytes and granulocytes to retard tumor outgrowth.

Transforming growth factor-beta1, a strong costimulator of rat T-cell activation promoting a shift towards a Th2-like cytokine profile.

TGF-beta is a known regulator of hematopoietic cells. In this study we suggest a major role of adherent spleen cells (adh-splc), to convert an inhibitory effect of TGF-beta1 on T-cell activation into a stimulatory effect. We show that interaction of TGF-beta1 with adh-splc induces a costimulatory effect on T-cell proliferation. This costimulatory signal requires the adh-splc to be in physical contact with the T-cells. Presence of adh-splc results in a shift towards a Th2-like response with a cytokine profile of increased IL-10 and decreased IFN-gamma. In the adh-splc population the increase of IL-10 is most pronounced at start of activation, whereas in the T-lymphocyte population, IL-10 increases at the end of culture. The suppression of the IFN-gamma production by TGF-beta1 is shown to be an important mechanism by which TGF-beta enhances proliferation of Th2 lymphocytes.

The three isoforms of transforming growth factor-beta co-stimulate rat T cells and inhibit lymphocyte apoptosis.

In this study the three different mammalian isoforms of transforming growth factor-beta (TGF-beta) were compared with regard to their effect on the response of rat T lymphocytes to the superantigen, staphylococcal enterotoxin A (SEA). All different isozymes were found to increase the proliferative response of rat T lymphocytes, which was accompanied by a significantly lower percentage of apoptotic cells than proliferation in the absence of TGF-beta. The same effect of TGF-beta was observed on the generation of apoptotic cells in an allo response (mixed lymphocyte reaction). TGF-beta2 and TGF-beta3 were three to 10-fold more potent than TGF-beta1 as co-stimulators of T lymphocytes, and equal in decreasing the percentage of apoptotic T cells. TGF-beta1 reduced the frequency and the number of cells undergoing apoptosis in T cells and, to an even higher degree, among B lymphocytes. TGF-beta did not seem to affect the production of the apoptosis inducer, tumour necrosis factor-alpha (TNF-alpha), neither at the mRNA level nor at the protein level. Neutralizing antibodies against the cytokine, TNF-alpha, decreased the percentage of apoptotic cells among T cells responding to SEA, both in the absence and in the presence of added TGF-beta1. Thus, when TGF-beta acts as a co-stimulator for T-cell activation it inhibits the induction of apoptosis and sustains the number of viable cells.

Monocyte-dependent costimulatory effect of TGF- beta 1 on rat T-cell activation.

TGF- beta 1 is known to have suppressive effects on both T-cell proliferation and effector functions, but costimulatory effects have also been reported. In the present investigation the effect of TGF- beta 1 is studied in vitro on T-cell proliferative responses of rat spleen cells and of lymph node cells to alloantigens (MLR), the superantigen Staphylococcal enterotoxin A (SEA) or IL-2. Without addition of TGF- beta 1, adherent, freshly isolated rat spleen monocytes have a suppressive effect on T-cell activation, which upon addition of TGF- beta 1 is reversed to a strong costimulatory effect. The costimulatory effect of TGF- beta 1 is shown to be entirely dependent on the presence of fresh monocytes. Costimulation is demonstrated when TGF- beta 1 is added to spleen cells at the start of the in vitro assays but not when added more than 24 h after the start. Costimulation is not demonstrable when TGF- beta 1 is added to lymph node cells alone but is readily detectable after admixture of freshly isolated spleen monocytes to the lymph node cells. TGF- beta 1 added at the end of culture induces suppression of T-cell activation irrespective of the presence or absence of monocytes. When TGF- beta 1 is added both at the start of an MLC and again after 4 days, the costimulatory effect is maintained, although somewhat moderated. The costimulatory effect of TGF- beta 1 is demonstrated as an increase of the T blast cell population of both CD4+ IL-2R+ and CD8+ IL-2R+ T-cell subsets, whereas the suppressive effect of TGF-beta 1 is shown as reduction of the same parameters.