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Insects are useful models for the study of innate immune mechanisms because of their lack of antibodies and receptors involved in adaptive immune response. Nevertheless, hemolin cloned from moths is a soluble and membrane associated Ig-related molecule that is up-regulated during immune response [Lanz-Mendoza, H. & Faye, I. (1999) Dev. Comp. Immunol. 23, 359-374]. The hemolin monomeric form has four, pair-wise, interacting Ig-domains, forming a strongly bent horseshoe structure [Su, X.-D., Gastinel, L.N., Vaughn, D.E., Faye, I., Poon, P. & Bjorkman, P. (1998) Science 281, 991-995]. To elucidate the nature of its homophilic and cellular interactions, the glycosylation and Ca2+-binding properties of hemolin were investigated. We used Hyalophora cecropia hemolin isolated from hemolymph of bacteria-injected pupae, or produced as a recombinant protein in a baculovirus/insect cell system. Both types of hemolin contain N-acetylglucosamine and probably sialic acid, as indicated by peptide:N-glycosidase F and neuraminidase digestion and glycosylation detection by Western-blotting analysis. The N-acetylglucosamine residues on hemolin were confirmed with the use of specific lectins. In addition, hemolin was shown to specifically bind calcium when spotted onto nitrocellulose and treated as for 45Ca2+ autoradiography. Earlier studies demonstrated that hemolin can bind to hemocytes and this was tested for its dependence on calcium and carbohydrates, using hemolin-coated fluorescent microspheres. A greater level of attachment of microspheres occurred in the presence of calcium than if calcium was absent. Furthermore, this binding was inhibited by EGTA and N-acetylglucosamine or N-acetylneuraminic acid, implying that carbohydrates and calcium are crucial factors in homophilic binding and cell-adhesion events mediated by this Ig-superfamily molecule.

The isolation of antibacterial peptides from the giant silkmoth Hyalophora cecropia has opened the area of animal antibiotics [Boman, H. G. (1991) Cell 65, 205-207] and the study of insect immune genes has revealed striking similarities to many immune response genes in mammals [Hultmark, D. (1994) Nature 267, 116-117]. However, the molecules and mechanisms behind primordial immune recognition are not understood. One candidate for one such recognition molecule is hemolin, a 48-kDa immunoglobulin-related protein first isolated from H. cecropia, where it is up-regulated upon infection and secreted into the hemolymph. Hemolin was shown to bind to bacteria and to hemocytes, giving rise to changes in hemocyte adhesiveness and intracellular phosphorylation patterns [Faye, I. & Kanost, M. (1997) in Molecular mechanisms of immune responses in insects (Brey, P. T. & Hultmark, D., eds) Chapman and Hall, London]. In the present publication, we give evidence for the presence of a 52-kDa membrane form of hemolin on hemocytes, based on flow-activated cell sorting and membrane protein extractions. In addition we reveal calcium-dependent homophilic binding properties of hemolin, using hemolin-coated microspheres. When biotinylated recombinant hemolin was allowed to bind to hemocyte membranes, higher molecular-mass complexes were formed. Furthermore, we used immunological methods and Northern-blot analysis to demonstrate the presence of hemolin in embryos and retinal discs, suggesting that hemolin is expressed in several tissues at different developmental stages. These results show novel cell adhesion features of hemolin, corroborating its multifunctional character with putative roles in cellular and humoral immunity and in development.

Insects have an effective humoral immune system, residing in immune proteins that are synthesized largely in the fatbody. kappa B-like motifs upstream of the immune protein genes bind the Cecropia immunoresponsive factor (CIF), and confer high levels of inducible expression. We have extended our studies and sought evidence that oxygen-derived active species might modulate the expression of the immune protein genes and the activation of CIF after the administration of different inducers. Mitogens, like arachidonic acid, phorbol esters, phytohemagglutinin, bacteria, and components of microbial cell envelopes stimulate expression of the attacin genes, both in vivo and in vitro. A general stimulant of oxidative stress, H2O2, stimulated expression of these genes and the weak immune response to wounding was greatly augmented by administration of H2O2. These responses were largely or entirely inhibited by dithiothreitol and by N-acetylcysteine. Nonspecific responses were excluded since immune genes failed to respond to albumin or starch, and the expression of a non-immunoresponsive gene was not affected. Electrophoretic mobility-shift assays showed that H2O2 and bacteria, when administered in vitro, could activate CIF in fatbody cells and that dithiothreitol and N-acetylcysteine prevented this process. Our data suggest that the induction of the immune protein genes is mediated through the activation of CIF, contingent upon thiol oxidation induced by oxidative stress.

Hemolin is an insect protein which belongs to the immunoglobulin superfamily and is strongly induced upon bacterial infection. It has been isolated from two moths, Hyalophora cecropia and Manduca sexta. We have isolated and sequenced a genomic clone for hemolin in H. cecropia, in order to resolve its organization and as a basis for investigating hemolin gene regulation. According to Southern-blot analysis, hemolin is encoded by a single gene, Hemolin. It contains six exons ranging over 32-603 bp. The introns are positioned both within and between the immunoglobulin-like domains, a feature typical for cell-adhesion molecules belonging to the immunoglobulin superfamily. By an RNase protection assay, we show that the Hemolin transcript is strongly induced not only by bacteria, but also by lipopolysaccharide and phorbol 12-myristate 13-acetate. Analysis of the upstream region and introns revealed potential binding sites for the Cecropia immunoresponsive factor (CIF), which recognizes the kappa B-like consensus GGGRA YYYYY.

The immune genes in Hyalophora cecropia contain an upstream sequence that is homologous to the binding site of the mammalian nuclear-factor kappa B (NF-kappa B). These genes are strongly induced by bacteria, lipopolysaccharides and 4 beta-phorbol 12-myristate 13-acetate. Induction of the immune genes involves the activation of a DNA-binding protein complex that we have named Cecropia immunoresponsive factor (CIF). CIF specifically recognizes the kappa B-like DNA sequences in the promoter regions of the Cecropia immune genes. The DNA binding activity of CIF correlates well with the transcriptional induction of the immune genes. Competition assays show that CIF has a DNA binding specificity similar to mammalian NF-kappa B. The two factors also share other characteristics, including the pattern of induction and the migration on the native gel.

To study the regulation of the immune genes in insects, we have cloned and sequenced the attacin gene locus of the giant silk moth Hyalophora cecropia. The locus contains one acidic and one basic attacin gene as well as two pseudogenes, which are remnants of basic attacin genes. A small insertion element was found within the locus. The two functional attacin genes are transcribed in opposite directions and have two introns inserted at homologous positions. A common sequence, GGGGATTCCT, is found at nucleotide position -48 in the acidic gene and at nucleotide position -58 in the basic gene. Interestingly, this decanucleotide is similar to the consensus of the NF-k B-binding site. Expression studies revealed that both attacins are strongly induced by phorbol 12-myristate 13-acetate, lipopolysaccharide and bacteria. However, only the acidic attacin gene showed a clear response to injury.

Pupae of the moth Hyalophora cecropia respond to an injection of live bacteria by the production of a potent antibacterial activity. The broad-spectrum property of this activity is due chiefly to two small proteins, cecropins A and B. Sequences of the proteins showed them to be homologous and to contain 37 and 35 amino acid residues respectively. The subsequent isolation of two cDNA clones for cecropin B showed that this protein is made as a prepro molecule composed of 62 amino acid residues. We have now prepared a genomic bank and studied four genomic clones for cecropin B. The coding regions were found in two neighbouring BglII fragments, one 0.79 kb and another varying in size from 3.1 kb to 4.9 kb for different clones. One transcriptional unit for preprocecropin B was sequenced and found to be 1035 bp long with a single intron, 514 bp in size. A conserved, insect specific cap site, ATCATTC, was identified by S1 mapping and primer extension experiments. Indications were found for the presence of multigene families and multicopy genes.