Bacterial heat shock proteins (HSPs) from Escherichia coli (GroES, GroEL, and DNAk) were tested for their ability to induce by themselves the expression and release of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-␣), and granulocyte-monocyte colony-stimulating factor (GM-CSF) by human monocytes and GM-CSF, IL-6, E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) by human umbilical vein endothelial cells (HUVEC). Our study demonstrated that treatment of monocytes with DNAk increased IL-6, TNF-␣, and GM-CSF release in a dose-dependent manner. The same effect was elicited by GroEL but at a lower rate. Treatment of HUVEC cultures with DNAk and GroEL also increased GM-CSF, IL-6, E-selectin, ICAM-1, and VCAM-1 release in a dose-dependent fashion. In any case, the greatest release was obtained by using DNAk and GroEL at a concentration of 1 g/ml. DNAk and GroEL were also able to up-regulate the surface expression of E-selectin, ICAM-1, and VCAM-1. As detected by reverse transcription-PCR analysis, DNAk and GroEL also increased the steady-state levels of cytokines and adhesion molecules in human monocytes and endothelial cells. In our study GroES showed a significant activity only on the release, surface expression, and mRNA transcription of E-selectin. Adhesion molecule expression seems to be a direct effect of HSPs and not via cytokines. Furthermore, these effects are due to HSPs properties because they are inhibited by specific monoclonal antibodies. These findings support the potential role of HSPs in modulating cell interactions during immunological and inflammatory responses. MATERIALS AND METHODS E. coli HSPs. E. coli HSPs GroES, the 10-kDa HSP (HSP 10), GroEL, the 60-kDa HSP (HSP 60), and DNAk, the 70-kDa HSP (HSP 70), were purchased from Boehringer Mannheim (Mannheim, Germany). Cell preparation and culture. (i) Monocyte isolation. To exclude variability, leukocyte populations were obtained for each test series from a buffy coat of a
Bacterial heat-shock proteins (HSPs) from Escherichia co/i (GroES, GroEL and DnaK) were studied for their ability to induce by themselves the expression and release of tumour necrosis factor-a (TNF-a), interleukin-I a (IL-1 a), interleukin-6 (IL-6) and intercellular adhesion molecule-I (ICAM-1) by cultured human keratinocytes. The surface expression of ICAM-1 was also investigated. In the supernatants of untreated cells none or a minimal amount of these molecules was found. After 48 h of stimulation with GroEL significant amounts of TNF-a, IGIa, IL-6 and soluble ICAM-I were detected, reaching maximum concentrations at 1 pg ml-l. The same effect was elicited by DnaK but to a lesser extent. Treatment of keratinocytes with GroEL and DnaK also increased TNF-a, IL-la, IL-6 and ICAM-I mRNA levels. GroES showed significant activity only on the expression and release of IL-6. GroEL and DnaK were also able to up-regulate the surface expression of ICAM-1 on keratinocytes. The effects on ICAM-1 expression seemed to be directly due to HSPs and not mediated via cytokines. Furthermore, these effects were due to the properties of HSPs because they were inhibited by specific monoclonal antibodies. These findings support the potential role of HSPs in modulating cell interactions during immunological and inflammatory responses in the skin.
In understanding the regulation of the specific immune response to Salmonella typhimurium, the role of a surface major component (porins) was studied. In this study we demonstrate that purified porins are able to induce a different response to that induced by the porins present on the S. typhimurium cell surface. Porin-treated or orally infected mice show anti-porin antibodies with bactericidal activity. The complete adoptive transfer of resistance to S. typhimurium is achieved only using splenic T cells from survivor mice after experimental infection. After stimulation with specific antigen in vitro CD4+ cells from porin-immunized mice released large amounts of interleukin-4 (IL-4), at a time when CD4+ cells from S. typhimurium-infected mice predominantly secreted interferon-gamma (IFN-gamma). Limiting dilution analysis showed that infection resulted in a higher precursor frequency of IFN-gamma-producing CD4+ T cells and a lower precursor frequency of IL-4-producing CD4+ T cells, while immunization with porins resulted in a higher precursor frequency of IL-4-producing cells and a low frequency of IFN-gamma-producing cells. Analysis of polymerase chain reaction-amplified cDNA from the spleens of infected mice revealed that IFN-gamma, IL-2 and IL-12 p40 mRNA were found 5 days after in vitro challenge and increased after 15 days; IL-10 expression was barely present after both 5 and 15 days, while IL-4 mRNA expression was not detected. In immunized mice, the IL-4 mRNA expression increased after 15 days, IFN-gamma mRNA expression disappeared entirely after 15 days, while IL-2, IL-10 and IL-12 mRNA remained relatively unchanged.
The response of Salmonella typhimurium to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 32-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Lipid and carbohydrate content of bacterial cells rapidly declined within the first 16 days, while DNA and proteins exhibited a more gradual decline over the 32 days of starvation. In contrast, RNA content did not decrease appreciably upon nutrient starvation. Structural damage occurred especially after 16 days of starvation. After 32 days of nutrient deprivation, we recorded degenerative cellular forms, a coccoidal cell shape, a decrease in cellular volume, and the loss of the three-layered outer membrane. The morphological and structural alterations correlated with virulence in infected animals. We observed a decrease in virulence of S. typhimurium after 9, 16, and 32 days of starvation, reaching a maximal decrease after 32 days of nutrient deprivation. The decrease in virulence correlated to surface hydrophobicity alterations, adherence to eukaryotic cells, and phagocytosis.
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