Leukotrienes do not regulate interleukin 1 production by activated macrophages.

The objective of this work was to investigate the role of leukotrienes in the production of IL-1 by activated human peripheral blood monocytes and mouse peritoneal macrophages. Using overnight adherent macrophages, stimulation with lipopolysaccharide or zymosan caused a time-dependent increase in IL-1 production. LTC4 was detected and preceded IL-1 production only in zymosan-treated macrophages. Lipopolysaccharide did not stimulate macrophages to produce LTC4. Zymosan-stimulated LTC4 production was inhibited by the lipoxygenase inhibitors, ICI207968 (3.20 microM), nordihydroguaiaretic acid (0.22 microM), phenidone (4.60 microM), REV5901 (0.20 microM), and the Merck 5-lipoxygenase "translocation inhibitor" MK886 (0.02 microM) with IC50 values as shown in parenthesis. However, none of these inhibitors reduced IL-1 production at concentrations which completely inhibited leukotriene synthesis. Taken together, these results do not support a role for leukotrienes in the production of IL-1 by zymosan-activated macrophages.

In vitro priming of human lymphocytes to heterologous insulins.

We have developed an in vitro priming assay in which peripheral blood lymphocytes from normal subjects are primed with insulin for 14 days prior to challenge with insulin in conjunction with autologous antigen-presenting cells for a further 5 days. Sheep, beef and pork insulins possess, respectively, four, three and one amino acid differences from the human molecule (out of a total of 51 residues) and the magnitude of the response to priming correlates with the degree of sequence variation. Although human insulin produces little response, priming with heterologous insulins readily induces auto-immunization on secondary challenge. The response to porcine priming was enhanced if the secondary cultures were challenged with bovine or ovine insulin, i.e., a heteroclitic response was observed. Individual donors differ in their response to priming and high responders possess the HLA-DR7 glycoprotein more frequently than low responders. This is in keeping with previous studies on antibody production in vivo and probably relates to the ease with which individual class II glycoproteins complex with processed antigen and stimulate T cells. This method has considerable potential for screening novel insulin molecules and formulations and should facilitate the mapping of helper and suppressor epitopes as well as the identification of agretopes involved in the presentation of insulin to T cells.

Identification of rat erythrocyte antigens with a new non-radioactive immunoprecipitation technique.

In order to examine how rat erythrocytes stimulate erythrocyte autoantibody production at the molecular level, we have identified rat erythrocyte antigens by immunoprecipitation and western blotting using monoclonal antibodies and antisera. A novel non-radioactive immunoprecipitation technique was used, which employed biotin as a label and a luminescent detection system. The new method was validated by comparison with conventional immunoprecipitation using 125I. Glycophorins of relative molecular mass (Mr) 81,000 and 38,000 were found to be the major antigenic components of rat erythrocytes, while band 3 (the most abundant erythrocyte membrane protein) was not recognized by rat-specific antibodies. The same surface antigens were recognized by sera from mice producing erythrocyte autoantibodies and by sera from mice in which autoantibody production was suppressed. Nine other minor rat-specific antigens were identified by blotting, ranging in Mr from 23,000 to 147,000. Analysis of the integral membrane proteins of rat and mouse erythrocytes by sodium dodecyl sulphate (SDS) electrophoresis followed by silver or periodic acid-Schiff (PAS) stains revealed differences between the glycophorins, but not between rat and mouse band 3. Thus, the major antigenic differences correspond to discernible biochemical differences between rat and mouse erythrocyte sialoglycoproteins.

Exofacial photoaffinity labelling of the human erythrocyte sugar transporter.

The 4-azidosalicylate derivative of 1,3-bis(D-mannos-4'-yloxy)-2-[2-3H]propylamine (ASA-[2-3H]BMPA) has been tested as a photoaffinity label for the sugar transporter in human erythrocytes. When photolysed in the presence of intact erythrocytes, ASA-[2-3H]BMPA covalently binds to the exofacial surface of the transporter. This labelled protein appears as a broad band in the 4.5 region in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The peak of radiolabel incorporation gives an apparent Mr of approx. 50 000 on 5-20% acrylamide gels. The binding is 80% inhibitable by 320 mM 4,6-O-ethylidene-D-glucose, by 320 mM D-glucose and by 50 microM cytochalasin B. Photoirradiation of a saturating concentration of ASA-BMPA in the presence of erythrocytes results in a 25-30% loss of D-galactose transport activity. From transport inactivation data and estimations of the amount of ASA-[2-3H]BMPA binding to the transporter it is calculated that there are approx. 220 000 exofacial hexose-transport binding sites per erythrocyte. The labelling of the transporter has been carried out using freshly drawn blood and 4-weeks-old transfusion blood. No change in the binding profile on SDS-polyacrylamide gel electrophoresis was observed. Proteolytic digestion of the ASA-[2-3H]BMPA-labelled transporter with either trypsin or alpha-chymotrypsin results in the appearance of a labelled 19 kDa fragment on SDS-polyacrylamide gel electrophoresis.

Interaction of hydrophobic bis (D-mannose) derivatives with adipocyte and erythrocyte sugar transport systems.

The inhibition of sugar uptake by a series of hydrophobic bis(D-mannose) derivatives has been measured in rat adipocytes. When the D-mannose moieties of the bis compounds are separated by a hexane bridge the transport inhibition constant (Ki) is greater than for a decane-bridged molecule. This is probably due to the increased hydrophobicity of the bridge of the decane-bridged compound. The enhancement in affinity due to the second sugar in the bis(D-mannose) derivatives is probably only 2-fold, since half reduction of the bis(D-mannosyloxy)hexane increases Ki approx. 2-3-fold. N'-DNP-1,3-bis(D-mannos-4'-yloxy)propyl-2-amine has very high affinity in insulin-treated cells. The affinity is approx. 1000-fold higher than for D-mannose. This enhancement is probably due to the hydrophobicity of the DNP group. The distance from the sugar to the hydrophobic group is important because an increase in Ki occurs if an aminocaproyl spacer is introduced between the DNP group and 1,3-bis(D-mannos-4'-yloxy)propyl-2-amine. Aminocaproyl and glycyl spacers also increase the Ki for NAP derivatives of 1,3-bis(D-mannos-4'-yloxy)propyl-2-amine. Each of the hydrophobic bis(D-mannose) derivatives has a lower Ki in insulin-treated cells. This may be due to an insulin responsive hydrophobic interaction between the hydrophobic portion of the sugar and a hydrophobic domain in the transport system. The inhibition constants for the hydrophobic bis(D-mannose) compounds have also been measured in human erythrocytes.

A new class of sugar analogues for use in the investigation of sugar transport.

D-Mannose derivatives have been synthesised which are crosslinked through their C-4 hydroxyls to propyl-2-amine. Coupling to the amino group gave a fluorodinitrobenzene derivative, a nitroazidophenyl derivative and an azidosalicylamide derivative. Each of these derivatives was shown to have high affinity for the human erythrocyte sugar transport system. The affinity constant for the nitroazidophenyl derivative was not altered by temperature changes. In rat adipocytes treated with insulin, the affinity constants for the derivatives were up to 1000-fold lower than for the parent sugar. In the absence of insulin the affinity constants for the derivatives, but not for D-mannose, were 3-times higher than in insulin-treated cells. By preparation of radiolabelled derivatives we have shown that the compounds are not transported either by erythrocytes or by adipocytes. Thus the crosslinked sugars are good outside-specific analogues.