The ability of interferon-alpha (IFN-alpha) to induce the adhesion molecules that characterize the islets of patients with type I diabetes has been investigated. We have found that all tested recombinant IFN-as will induce major histocompatibility complex (MHC) class I on arterial endothelial cells. Some but not all IFN-as will induce intercellular adhesion molecule-1 (ICAM-1). However, there is only a transient and modest increase in VCAM on arterial endothelial cells. IFN-alpha has very little effect on endothelial MHC class II expression but will induce these proteins on monocytes. Thus, there is a close concordance between the biological actions of IFN-alpha and the appearance of those adhesion molecules induced in the islets of patients with type I diabetes. IFN-alpha is also produced in normal human islets during short-term cultures, probably as a result of the ischemia present at the center of the islet. This induction of IFN-alpha by hypoxia may explain the previously reported spontaneous induction of ICAM-1 in human islets and may also be a contributing factor to the failure of islet grafts.

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To determine why Ld antigens are expressed on the cell surface at levels three to four times lower than Dd or Kd antigens, pulse-chase experiments were used to compare their rates of biosynthesis and processing. Electrophoresis on sodium dodecyl sulfate gradient polyacrylamide gels resolved immunoprecipitates of each of these histocompatibility complex class I molecules into a slower and faster species. During the chase period, the faster migrating species appeared to be converted to the slower migrating species in a time-dependent manner. However, the conversion of Ld from the faster to the slower migrating species proceeded significantly more slowly than did the conversion of either Dd or Kd. Endoglycosidase H sensitivity and cell surface radiolabeling were used to determine the glycosylation state and cell location of each species of Ld and Dd. The results from these experiments, along with the pulse-chase studies and cytofluorometric analyses, suggest that Ld possesses a much slower rate of processing from a faster migrating, high mannose-bearing species to a slower migrating, complex oligosaccharide-bearing species found on the cell surface. Analysis of the beta 2-microglobulin (beta 2-m) association confirmed that Ld is associated with less beta 2-m than Dd. To localize the structures on class I molecules influencing their surface expression, rate of processing, and beta 2-m association, the Ddm1 molecule was analyzed. The Ddm1 molecule of the mutant B10.D2-H-2dm1 has previously been shown to be a chimeric Dd (amino-terminal)/Ld (carboxyl-terminal) polypeptide. The surface expression, processing and beta 2-m association of Ddm1 were found to be similar to Dd rather than Ld, suggesting that each of these phenomena are influenced by protein structure in the amino terminus.

We have performed a structure-function analysis of extracellular domain regions of the human IFN-α receptor (hIFNAR1) using mAbs generated by immunizing mice with a soluble hIFNAR1-IgG. Five mAbs described in this study recognize different epitopes as determined by a competitive binding ELISA and by alanine substitution mutant analyses of the hIFNAR1-IgG. Two mAbs, 2E1 and 4A7, are able to block IFN-stimulated gene factor 3 (ISGF3) formation and inhibit the antiviral cytopathic effect induced by several IFN-α (IFN-α2/1, -α1, -α2, -α5, and -α8). None of these anti-IFNAR1 mAbs were able to block activity of IFN-β. mAb 4A7 binds to a domain 1-hIFNAR1-IgG but not to a domain 2-hIFNAR1-IgG, which suggests that its binding region is located in domain 1. The binding of the most potent blocking mAb, 2E1, requires the presence of domain 1 and domain 2. The most critical residue for 2E1 binding is a lysine residue at position 249, which is in domain 2. These findings suggest that both domain 1 and domain 2 are necessary to form a functional receptor and that a region in domain 2 is important. IFN-β recognizes regions of the hIFNAR complex that are distinct from those important for the IFN-α.