Immune Class Regulation and Its Medical Significance Part II of a Report of a Workshop on Foundational Concepts of Immune Regulation.

We discussed different proposals for how the nature of the Th1/Th2 phenotype of an immune response is determined, and favoured one, the Threshold Hypothesis, as plausible and so useful as the basis for further discussions. The activation of a target CD4 T cell can be facilitated by helper CD4 T cells when the CD4 T cells interact via an antigen-presenting cell. The Threshold Hypothesis states that tentative and robust antigen-mediated CD4 T cell cooperation results in the target CD4 T cell, respectively giving rise, upon activation, to Th1 and Th2 cells. We primarily discussed four topics. We briefly discussed in the background section certain limitations of the Th1/Th2 paradigm in understanding immune class regulation, and the remarkable anti-inflammatory properties of human IgG4 antibody. Secondly, we assessed the role of class II MHC molecules in determining the number of mature CD4 T cells and so affecting the Th1/Th2 phenotype of immune responses. We also discussed the controversial role of CD8 T cells in affecting the Th1/Th2 phenotype of responses to MHC and other antigens, and the potential role of their relative scarcity in neonates in biasing responses towards an antibody, Th2 mode. Lastly, we examined the regulation of the Th1/Th2 phenotype of both primary and ongoing immune responses in the context of the intriguing proposal that antigen initially generates different classes/subclasses of immunity and then selects, by a feedback mechanism, the most effective class. We found this interesting idea difficult to reconcile with various observations.

Immunological Tolerance. Part I of a Report of a Workshop on Foundational Concepts of Immune Regulation.

This report, the first of two, arose from a one-week workshop directed at discussing concepts of immune regulation, and focuses on immunological tolerance. We first outline the major ideas we thought sufficiently plausible to provide a context for discussing more controversial issues around tolerance. We then report on our discussion of different experiments that appear paradoxical in terms of the different, contemporary models of CD4 T cell inactivation/activation, and how such observations might be resolved in terms of insights provided by these contemporary models. These discussions bear on the plausibility of the Pathogen-Associated Molecular Pattern (PAMP), Danger and Two Step, Two Signal Models for the activation of naïve CD4 T cells. Some of the observations considered appear paradoxical in terms of the PAMP and Danger Models, but not with the Two Step, Two Signal Model. For example, genetically immunodeficient mice have been given foreign, sterile ectopic grafts, and the immune system allowed to develop once these grafts were well-healed in, and so in the absence of PAMPs or danger. The grafts were rejected, unexpected on the PAMP or Danger Models. We also discussed considerations and observations bearing on the widely held idea that antigen must crosslink the membrane Ig receptors of a B cell to initiate the generation of signal 1, or the alternative possibility that monovalent binding by antigen can do so. We favored the latter possibility, and discussed a particular model, "the Elbow Model," for how this might be achieved.

Generation of antibody-producing hybridomas following one single immunization with a targeted DNA vaccine.

The standard protocol for generating antibody (Ab)-producing hybridomas is based on fusion of plasmacytoma cells with Ab-producing B cells harvested from immunized mice. To increase the yield of hybridomas, it is important to use immunization protocols that induce a high frequency of B cells producing specific Abs. Our laboratory has developed a vaccine format, denoted vaccibody that promotes the immune responses towards the delivered antigen. The vaccine format targets antigens in a bivalent form to surface receptors on antigen-presenting cells (APCs). Here, we used the fluorescent protein (FP) mCherry as antigen and targeted it to APCs by use of either the natural ligand CCL3/MIP-1α or single-chain variable fragment specific for major histocompatibility complex class II. The vaccine format was delivered to mouse muscle as DNA combined with electroporation. By this procedure, we developed two monoclonal Abs that can be utilized to detect the FC mCherry in various applications. The data suggest that the targeted DNA vaccine format can be utilized to enhance the number of Ab-producing hybridomas and thereby be a tool to improve the B cell hybridoma technology.

Specific endocytosis and catabolism in the scavenger endothelial cells of cod (Gadus morhua L.) generate high-energy metabolites.

The catabolic fate of circulating hyaluronan and the proteoglycan chondroitin sulphate (CSPG) was studied in the Atlantic cod (Gadus morhua L.). Distribution studies using radio-iodinated ligand demonstrated that CSPG was rapidly eliminated from the blood by the endocardial endothelial cells (EECs) of the heart atrium and ventricle. The presence of excess amounts of hyaluronan or CSPG inhibited uptake of [125I]hyaluronan into cultured atrial EECs (aEECs) by 46% and 84%, respectively. Neither formaldehyde-treated serum albumin (FSA) nor mannose inhibited this uptake. The presence of excess amounts of CSPG and hyaluronan inhibited uptake of [125I]CSPG by 90% and 42%, respectively, suggesting that aEECs express a specific hyaluronan binding site that also recognizes CSPG. FSA inhibited endocytosis of [1251]CSPG by 65%, indicating that CSPG is also recognized by the scavenger receptor. Approximately 17% and 57% of added [125I]hyaluronan and 15% and 65% of the added [125I]CSPG were endocytosed after 1 and 24h, respectively. High-performance liquid chromatographic analyses of the spent medium after endocytosis of hyaluronan and CSPG serglycin labelled biosynthetically with 3H in the acetyl groups identified labelled the low-molecular-mass degradation products as [3H]acetate, indicating that aEECs operate anaerobically. These findings suggest that acetate released from cod EECs following catabolism of endocytosed hyaluronan and CSPG represents a high-energy metabolite that may fuel cardiomyocytes.

Serglycin secreted by leukocytes is efficiently eliminated from the circulation by sinusoidal scavenger endothelial cells in the liver.

This study was undertaken to determine the fate of the circulating chondroitin sulfate proteoglycan serglycin. The human monocytic cell line THP-1 was cultured under serum-free conditions in the presence of [35S]sulfate. The conditioned medium was harvested and 35S-macromolecules were purified by Q-Sepharose anion-exchange chromatography and Superose 6 gel chromatography. After labeling with 125I, the purified material was treated with chondroitinase ABC and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. A major band with mr of approximately 14 kDa appeared, consistent with the core protein of serglycin. The identity of the proteoglycan was confirmed by amino-terminal amino acid sequencing. Purified serglycin, labeled either with [35S]sulfate or 125I and fluorescein isothiocyanate, was injected intravenously into rats. The blood content of radiolabeled serglycin fell by 50% from 1 to 2.4 min after injection, indicating an initial t1/2 of 1.4 min or shorter. Approximately 90% of the recovered radioactivity was localized in the liver, 5% in the blood, and 5% altogether in urine, kidneys, and spleen about 30 min after injection. Isolation of liver cells at the same time point showed that 70% of the radioactivity was taken up by the sinusoidal scavenger endothelial cells, and 23 and 7% by the hepatocytes and Kupffer cells, respectively. When excess amounts of unlabeled hyaluronan was coinjected with radiolabeled serglycin, the elimination of serglycin was significantly inhibited, indicating that the hyaluronan receptor on the sinusoidal scavenger endothelial cells is responsible for the elimination of serglycin.