The location ofC2, C4, andBF relative toHLA-B andHLA-D

The loci forHLA-A, B, C, D, andDR are known to be closely linked to the structural loci for the complement components C2, BF, and the duplicated loci for C4, C4A and C4B. Conflicting evidence has been presented for the order of these genes. However, new techniques have made possible identification of markers in theHLA-D andC4 region for nearly all identified haplotypes. In our population we have confirmed fiveHLA-B-D crossovers and in each case informative allotypes of C2, BF, or C4A and C4B segregated withHLA-D orDR suggesting that the loci for these proteins lie close toHLA-D andDR. These findings may be of importance for resolving problems encountered in the assignment ofHLA-D alleles.     

Linkage of C4 and C4 deficiency to BF and GPLA

The C4, Bf, and GPLA phenotypes of homo- and heterozygous C4-deficient guinea pigs were studied. The electrophoretic patterns suggest that the deficiency in circulating C4 results from an impaired structural gene, allelic to the C4^F, C4^S, and C4^S1 alleles at the C4 locus. In family studies, support for linkage of C4 and Bf to theGPLA system was obtained. The defective gene appears to be the fourth allele, which is rare, in the polymorphism of the fourth component of guinea pig complement.Abbreviations used in this paper are as follows Bf locus for properdin factor B - MHC major histocompatibility complex - GPLA major histocompatibility complex of the guinea pig     

Partial C4 deficiency in subacute sclerosing panencephalitis

In an immunogenetic study, 23 subacute sclerosing panencephalitis (SSPE) patients and their families were studied for the HLA region markers HLA-A, B, C, DR, BF, C2, C4A, C4B, GLO I, and PGM3. In addition, C3, C4, and factor B serum levels were determined. A highly significant association of C4A^*QO with SSPE was found. Furthermore, two rare haplotypes, C4A*QOB*9QO, two C4ACh+ allotypes, and four Ch partial inhibitors were detected, which possibly impair the function of the C4 molecules. HLA-DR5 was increased. In addition, a number of rare HLA-A, C, B, DR haplotypes were observed. It is postulated that rare C4 molecular deficiency might be a predisposing factor in the pathogenesis of SSPE.     

Genetics of human C4 polymorphism: Detection and segregation of rare and duplicated haplotypes

Applying a combined technology for the detection of allotypec variation of the fourth component of human complement (C4), including immunofixation with anti-C4 and C4-dependent lysis after agarose electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of C4 to separate the C4A and B -chains, and the determination of Rodgers (Rg) and Chido (Ch) determinants of C4 in serum and at the blotted C4 -chains, we detected rare human C4 allotypes and studied the genetic linkage. Partial inhibitors (p. i.) of anti-Rg and anti-Ch sera were found; the C4A51 allotype characterized as Rg p. i. and the C4A1 and C4B51 allotypes as Ch p. i. were genetically inherited. The C4A1 allotype has a unique Rg^- Ch⁺ C4A -chain. Duplicated C4A loci, A ^*3, A ^*2, and A ^*5, A ^*2 were both associated with a C4BQO and the HLA haplotype A3-Cw4-Bw35-DR1. These additions to the already known extensive C4 polymorphism may help to sort out their significance for the biological functions of human C4.Abbreviations used in this paper BF Factor B polymorphism of the alternative pathway of complement activation - C2 second component of complement - C4 fourth component of complement - C4D C4-deficient (C4^*QO/QO) - Ch Chido determinant on C4B^* products - EDTA ethylendiaminetetraacetic acid - GLO I glyoxalase I - HLA human leucocyte antigens, A, B, C and DR (D =related) loci - PAGE polyacrylamide gel electrophoresis - PGM3 phosphoglucomutase, third locus - p. i. partial inhibitor = serological inhibition of some, but not all anti-Ch and anti-Rg sera at selected dilutions - SDS sodium dodecyl sulphate; 94k/96k, 94 000 and 96 000 dalton molecular weight Presented in part at the 1V International Workshop on the Genetics of Complement, July 13–15, 1982, Boston, MA, and the Xth International Complement Workshop, May 25–27,1983 in Mainz, Federal Republic of Germany.     

Heterogeneity of human C4 gene size

In this article we present a study showing that the human C4 genes differ in length because of the presence or absence of a 6.5 kb intron near the 5 end of the gene. DNA from individuals of known HLA, factor B, and C4 haplotypes was analyzed for restriction fragment length polymorphism (RFLP) by Southern blot analysis with C4-specific cDNA probes. The RFLP patterns obtained showed that the C4 genes are either 22.5 kb or 16 kb in length. They are referred to as long and short C4 genes, respectively. A population study was carried out to examine the distribution of the gene size according to C4 allotypes and haplotypes. Long C4 genes included all C4A genes studied and also some C4B allotypes, e. g., B1 on most C4 A3B1 haplotypes. Similarly, C4B null genes were found to be of the long form. Other C4B allotypes tested were found to be coded for by short C4 genes, including B2, B1 in C4 A6B1 and C4 AQOB1 (with a single C4B gene haplotype).Abbreviations used in this paper C4 fourth component of complement - C2 second component of complement - BF factor B - MHC major histocompatibility complex - RFLP restriction fragment length polymorphism - EDTA ethylenediaminetetraacetic acid - SDS lauryl sulfate, sodium salt     

C4 urernic variant: An acquired C4 allotype

The major histocompatibility complex (MHC)-linked complotype region includes alleles for B, C2, and C4 loci. These loci are closely linked to each other and to HLA-DR on chromosome 6. The duplicated C4 loci,, C4A and B, are especially polymorphic. In seven patients with renal insufficiency, we observed a C4 variant with electrophoretic mobility between C4B2 and C4B3. Four of these patients were detected during a study of MHC markers in mernbranoproliferative glomerulonephritis. Complete complotype and HLA data from families of five of the seven patients demonstrated that the variant was not inherited. The pattern was revealed by immunofixation electrophoresis and also by C4-specific hemolytic overlay. In serial plasma specimens taken from one patient, the C4 variant appeared only after the patient became uremic. However, the variant could not be produced in normal plasma after incubation with C4-depleted uremic plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions of immunoprecipitated C4 from these patients showed C4A and C4B chains of normal molecular mass; incompletely processed forms of C4 were not observed. We believe that this variant is probably acquired in the presence of uremia and may represent the C4B2.9 allele found by Wank and co-workers in many patients with glomerulonephritis. Family studies are mandatory to distinguish genetic variants from acquired alterations in the C4 phenotype.     

C6-Like and C3-Like Molecules from the Cephalochordate, Amphioxus, Suggest a Cytolytic Complement System in Invertebrates

The mammalian immune system has cytotoxic mechanisms, both cellular and humoral, that destroy the membrane integrity of target cells. The main effector molecules of these cytolytic mechanisms—perforin, used by killer lymphocytes, and the membrane attack complex (MAC) components of the complement system—share a unique module called the MAC/perforin module. Until now, both immunological cytotoxicity and the MAC/perforin module have been reported only in jawed vertebrates. Here, we report the identification of a protein containing the MAC/perforin module from the invertebrate cephalochordate, amphioxus (Branchiostoma belcheri), using expressed sequence tag (EST) analysis of the notochord. The deduced amino acid sequence of this molecule is most similar to the primary structure of human complement component C6 and is designated AmphiC6. AmphiC6 shares a unique modular structure, including the MAC/perforin module, with human C6 and other MAC components. Another EST clone predicts the presence of a thioester-containing protein with the closest structural similarity to vertebrate C3 (therefore designated AmphiC3). AmphiC3 retains most of the functionally important residues of vertebrate C3 and is shown by phylogenetic analysis to be derived directly from the common ancestor of vertebrate C3, C4, and C5. Only opsonic activity has been assigned to the invertebrate complement system until now. Therefore, this is the first molecular evidence for complement-mediated immunological cytotoxicity in invertebrates. Received: 24 August 2001 / Accepted: 12 November 2001     

GroEL1, a Heat Shock protein 60 of Chlamydia pneumoniae,Impairs Neovascularization by Decreasing Endothelial Progenitor Cell Function

The number and function of endothelial progenitor cells (EPCs) are sensitive to hyperglycemia, hypertension, and smoking in humans, which are also associated with the development of atherosclerosis. GroEL1 from Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis. However, the actual effects of GroEL1 on EPC function are unclear. In this study, we investigate the EPC function in GroEL1-administered hind limb-ischemic C57BL/B6 and C57BL/10ScNJ (a toll-like receptor 4 (TLR4) mutation) mice and human EPCs. In mice, laser Doppler imaging, flow cytometry, and immunohistochemistry were used to evaluate the degree of neo-vasculogenesis, circulating level of EPCs, and expression of CD34, vWF, and endothelial nitric oxide synthase (eNOS) in vessels. Blood flow in the ischemic limb was significantly impaired in C57BL/B6 but not C57BL/10ScNJ mice treated with GroEL1. Circulating EPCs were also decreased after GroEL1 administration in C57BL/B6 mice. Additionally, GroEL1 inhibited the expression of CD34 and eNOS in C57BL/B6 ischemic muscle. In vitro, GroEL1 impaired the capacity of differentiation, mobilization, tube formation, and migration of EPCs. GroEL1 increased senescence, which was mediated by caspases, p38 MAPK, and ERK1/2 signaling in EPCs. Furthermore, GroEL1 decreased integrin and E-selectin expression and induced inflammatory responses in EPCs. In conclusion, these findings suggest that TLR4 and impaired NO-related mechanisms could contribute to the reduced number and functional activity of EPCs in the presence of GroEL1 from C. pneumoniae.     

Quantitative variation of C4 variant proteins associated with many MHC haplotypes

C4 protein variants were analyzed in 64 individuals, of which 51 were either homozygous or heterozygous for an extended major histocompatibility complex (MHC) haplotype (a fixed combination of MHC alleles). The relative amount of each C4 variant was measured by densitometric scanning of stained immunofixed electrophoretic patterns of neuraminidase- and carboxypeptidase-treated samples. The relative concentrations of C4 variants on any haplotype were stable and inherited in families. In five of the eight extended haplotypes investigated, the amount of one of the C4 variants relative to others in the same pattern was increased:[HLA-B8, SC01, DR3] and[HLA-B7, SC31, DR2] produced an approximately doubled amount of C4B1;[HLA-B18, S042, DR2] an increased amount of C4B2; and[HLA-B44, SC30, DR4] a double amount of C4A3. The extended haplotype[HLA-Bw57, SC61, DR7] gave rise to two to three times as much C4B1 as C4A6. In the extended haplotypes[HLA-B44, FC31, DR7] and[HLA-Bw62, SC33, DR4], the results did not clearly indicate differences in expression of the C4 isotypes. DNA analysis possibly supported an actual gene duplication only for the haplotype[HLA-B7, SC31, DR2]. The results suggest that, in addition to variation in the number of structural genes, other MHC-linked mechanisms may be involved in the regulation of the relative amounts of C4A or C4B protein specified by any haplotype.     

Binding of the complement inhibitor C4bp to serogroup B Neisseria meningitidis

Neisseria meningitidis (meningococcus) is an important cause of meningitis and sepsis. Currently, there is no effective vaccine against serogroup B meningococcal infection. Host defense against neisseriae requires the complement system (C) as indicated by the fact that individuals deficient in properdin or late C components (C6-9) have an increased susceptibility to recurrent neisserial infections. Because the classical pathway (CP) is required to initiate efficient complement activation on neisseriae, meningococci should be able to evade it to cause disease. To test this hypothesis, we studied the interactions of meningococci with the major CP inhibitor C4b-binding protein (C4bp). We tested C4bp binding to wild-type group B meningococcus strain (H44/76) and to 11 isogenic mutants thereof that differed in capsule expression, lipo-oligosaccharide sialylation, and/or expression of either porin (Por) A or PorB3. All strains expressing PorA bound radiolabeled C4bp, whereas the strains lacking PorA bound significantly less C4bp. Increased binding was observed under hypotonic conditions. Deleting PorB3 did not influence C4bp binding, but the presence of polysialic acid capsule reduced C4bp binding by 50%. Bound C4bp remained functionally active in that it promoted the inactivation of C4b by factor I. PorA-expressing strains were also more resistant to C lysis than PorA-negative strains in a serum bactericidal assay. Binding of C4bp thus helps Neisseria meningitidis to escape CP complement activation.     

HLA Haplotypes with C4B5; evidence for further allelic heterogeneity

Twenty-three individuals from various disease groups and normal controls were identified by immunofixation with anti-C4, C4-dependent lysis, determination of Rg (Rodgers) and Ch (Chido) phenotypes, and immunoblotting with C4-specific mouse monoclonal antibody. We found that one haplotype predominates with the C4B ^* 5 allele, HLA-A11, B22(55), Cw3, Bf ^* S, C4A ^* 4B ^* 5, which also carries the Ch ^{1,–2, 3} haplotype. The B5 allotype was also found with HLA-1360, HLA-1335 in Caucasoids, and HLA-B18 in non-Caucasoids; these carried the Ch ^{–1, –2, –3} haplotype. Our results are in accord with an earlier report of two B5 subtypes, B5Rg⁺ and B5Rg^– (Roos et al. 1984). The specificity of the mouse monoclonal antibodies IC4 and 21312 had been previously related to C4A and C4B, respectively, but our results suggest that they relate more closely to Rg and Ch determinants.     

The C-terminal domains of human TNRC6A,TNRC6B,and TNRC6C silence bound transcripts independently of Argonaute proteins

Proteins of the GW182 family are essential components of the miRNA pathway in animal cells. Vertebrate genomes encode three GW182 paralogs (TNRC6A, TNRC6B, and TNRC6C), which may be functionally redundant. Here, we show that the N-terminal GW-repeat-containing regions of all three TNRC6s interact with the four human Argonaute proteins (AGO1–AGO4). We also show that TNRC6A, TNRC6B, and TNRC6C silence the expression of bound mRNAs. This activity is mediated by their C-terminal silencing domains, and thus, is independent of the interaction with AGO1–AGO4. Silencing by TNRC6A, TNRC6B, and TNRC6C is effected by changes in protein expression and mRNA stability that can, in part, be attributed to deadenylation. Our findings indicate that TNRC6A, TNRC6B, and TNRC6C are recruited to miRNA targets through an interaction between their N-terminal domain and an Argonaute protein; the TNRC6s then promote translational repression and/or degradation of miRNA targets through a C-terminal silencing domain.     

C4 allotyping distinguishes HLA-1314.1 and B14.2 subgroups

Complement allotyping (C4, C2, and BF) was performed in 60 unrelated individuals and 15 families characterized for the subtypes (14.1 and 14.2) within HLA-B14. Eighty-seven percent of B14.2 individuals typed positive for the rare C4A2 variant. In contrast, less than 7% of B14.1 individuals were positive for this C4 allotype which is in keeping with a control background frequency. Family studies revealed that three distinct complotypes (complement haplotypes) are characteristic for the two HLA-1314 subgroups. The SC22 and FC31 complotypes characterize the B14.2 subtype, whereas SC31 appears to define B14.1.     

The molecular basis for the difference in immune hemolysis activity of the Chido and Rodgers isotypes of human complement component C4

Human C4 displays a structural polymorphism which is consistent with there being two closely linked genetic loci coding for this protein. These give rise to two C4 isotypes, designated C4A and C4B, which can be distinguished by charge and apparent m.w. differences in their respective alpha-chains and by the presence or absence of the Chido/Rodgers blood group antigens. Previous qualitative studies of C4 immune hemolysis activity in whole plasma had suggested that the C4B isotype was functionally more active. By using purified C4A and C4B isolated from individual donors known serologically to possess only one of the C4 isotypes, we examined the molecular basis for the differences in their respective hemolytic activities. It was found that the C4B:C4A hemolytic activity ratio was approximately 4:1. This fourfold difference could not be accounted for by a commensurate difference in the cleavage rate of the two isotypes by C1s by differences in the kinetics of assembly or intrinsic decay of the respective C3 convertase enzymes, or by differences in the rate of isotypic C4b cleavage by factor I in the presence of C4bp . However, the fourfold greater deposition efficiency of nascent C4b of the C4B isotype onto the surface of C1-bearing sheep erythrocytes quantitatively accounted for the observed difference in immune hemolysis function. It was further found that the thioester bond of nascent C4b of the C4A isotype preferentially transacylates onto amino group nucleophiles, whereas in the C4B isotype, acylation of hydroxyl groups is strongly preferred. Thus, the difference in immune hemolysis activity between the two C4 isotypes does not necessarily indicate an impairment of function in C4A; it may merely be a reflection of the relative abundance at the surface of a C1-bearing target of hydroxyl and amino groups capable of being acyl acceptors for nascent C4b. Finally, we also present evidence showing that the apparent m.w. difference between the alpha-chains of the C4A and C4B isotypes is not due to differences in protein glycosylation.     

Structure and dynamics of the N-terminal half of hepatitis C virus core protein: an intrinsically unstructured protein

It has been reported that cyclosporine A (CsA) aggravates vascular injury in hyperlipidemic patients, but the specific mechanisms are unclear. We explored the hypothesis that CsA may result in complement-mediated endothelial cell lysis induced by down-regulation of decay-accelerating factor (DAF) in hyperlipidemic patients. Human umbilical vein endothelial cells (HUVECs) were treated with CsA or/and oxidized low-density lipoprotein (ox-LDL) before allowing DAF expression. Complement factor C3 cell binding was measured by flow cytometry. CsA exposure led to decreased DAF expression and aggravated cell lysis of the HUVECs pre-incubated with ox-LDL, in a dose-dependent fashion. In in vivo experiments using thoracic aortic endothelium from hyperlipidemic rats, CsA resulted in dose-dependent down-regulation of DAF, and accompanying endothelial damage. These observations provide new evidence that hyperlipidemic patients treated with CsA may have an increased vascular risk, at least in part through complement-mediated EC lysis following down-regulated DAF expression.     

Complete complement components C4A and C4B deficiencies in human kidney diseases and systemic lupus erythematosus

Although a heterozygous deficiency of either complement component C4A or C4B is common, and each has a frequency of approximately 20% in a Caucasian population, complete deficiencies of both C4A and C4B proteins are extremely rare. In this paper the clinical courses for seven complete C4 deficiency patients are described in detail, and the molecular defects for complete C4 deficiencies are elucidated. Three patients with homozygous HLA A24 Cw7 B38 DR13 had systemic lupus erythematosus, mesangial glomerulonephritis, and severe skin lesions or membranous nephropathy. Immunofixation, genomic restriction fragment length polymorphisms, and pulsed field gel electrophoresis experiments revealed the presence of monomodular RP-C4-CYP21-TNX (RCCX) modules, each containing a solitary, long C4A mutant gene. Sequencing of the mutant C4A genes revealed a 2-bp, GT deletion in exon 13 that leads to protein truncation. The other four patients with homozygous HLA A30 B18 DR7 had SLE, severe kidney disorders including mesangial or membranoproliferative glomerulonephritis, and/or Henoch Schoenlein purpura. Molecular genetic analyses revealed an unusual RCCX structure with two short C4B mutant genes, each followed by an intact gene for steroid 21-hydroxylase. Nine identical, intronic mutations were found in each mutant C4B. In particular, the 8127 g-->a mutation present at the donor site of intron 28 may cause an RNA splice defect. Analyses of 12 complete C4 deficiency patients revealed two hot spots of deleterious mutations: one is located at exon 13, the others within a 2.6-kb genomic region spanning exons 20-29. Screening of these mutations may facilitate epidemiologic studies of C4 in infectious, autoimmune, and kidney diseases.     

Autophagy,proteases and the sense of balance

The knowledge of the molecular mechanisms underlying autophagy has considerably improved after the isolation and characterization of autophagy-defective mutants in the yeast Saccharomyces cerevisiae. Two ubiquitin-like conjugation systems are required for yeast autophagy. One of them requires the participation of Atg8 synthesized as a precursor protein, which is cleaved after a Gly residue by a cysteine proteinase called Atg4. The new Gly-terminal residue from Atg8 is activated by Atg7 (an E1-like enzyme) then transferred to Atg3 (an E2-like enzyme) and finally conjugated with membrane-bound phosphatidylethanolamine (PE) through an amide bond. The complex Atg8–PE is also deconjugated by the protease Atg4, facilitating the release of Atg8 from membranes. This modification system, which is essential for the membrane rearrangement dynamics that accompany the initiation and execution of autophagy, is conserved in higher eukaryotes including mammals. We have previously identified and cloned the four human orthologues of the yeast proteinase Atg4, whereas parallel studies have revealed that there are at least six orthologues of yeast Atg8 in mammals (LC3A, LC3B, LC3C, GABARAP, ATG8L/GABARAPL1 and GATE-16/GABARAPL2). Thus, in mammals, the Atg4-Atg8 proteolytic system is composed of four proteinases (autophagins) that may target at least six distinct substrates, contrasting with the simplified yeast system in which one single protease cleaves a sole substrate. Currently, it is unclear why mammals have developed this array of closely related enzymes, as other essential autophagy genes such as Atg3, Atg5 or Atg7 are represented in mammalian cells by a single orthologue. It has been suggested that the multiplication of Atg4 orthologues may reflect a regulatory heterogeneity of functionally redundant proteins or, alternatively, derive from the acquisition of new functions that are not related to autophagy. Our first approach to elucidate this question was based on the generation of autophagin-3/Atg4C-deficient mice, which however presented a minor phenotype. With the generation of autophagin-1/Atg4B-deficient mice, recently reported, we have progressed in our attempt to identify the in vivo physiological and pathological roles of autophagins.     

Induced Expression of Cathepsins and Cystatin C in a Murine Model of Demyelination

While proteolytic enzymes are involved in the pathogenesis of multiple sclerosis (MS), the involvement of cathepsins has not been characterized in detail. To better understand the role of cathepsins, cDNA microarray analysis was used to study the brains of proteolipid protein transgenic (plp ^tg /−) mice, an animal model that closely mimics the failure of remyelination in MS. Analysis revealed upregulated expression of cathepsins L, H and B and their inhibitor, cystatin C. By in situ hybridization, the induction of cathepsins was primarily limited to microglia/macrophages of the white matter, with continuous expression from 2 to 8 months of age. Elevated protein level of cathepsins was confirmed at 4 months of age. In contrast, elevated expression of cystatin C was found in astrocytes. The ratio of microglia/macrophages to astrocytes increased throughout the course of demyelination, suggesting that the ratio of secreted cathepsins to cystatin C increased during that period. We propose that in MS, remyelination may be impaired by increasing activity of cathepsins inadequately controlled by cystatin C. Special issue dedicated to Anthony Campagnoni.     

Palmitate-induced Down-regulation of Sortilin and Impaired GLUT4 Trafficking in C2C12 Myotubes

Elevated saturated FFAs including palmitate (C16:0) are a primary trigger for peripheral insulin resistance characterized by impaired glucose uptake/disposal in skeletal muscle, resulting from impaired GLUT4 translocation in response to insulin. We herein demonstrate that palmitate induces down-regulation of sortilin, a sorting receptor implicated in the formation of insulin-responsive GLUT4 vesicles, via mechanisms involving PKCθ and TNF-α-converting enzyme, but not p38, JNK, or mitochondrial reactive oxygen species generation, leading to impaired GLUT4 trafficking in C2C12 myotubes. Intriguingly, unsaturated FFAs such as palmitoleate (C16:1) and oleate (C18:1) had no such detrimental effects, appearing instead to effectively reverse palmitate-induced impairment of insulin-responsive GLUT4 recycling along with restoration of sortilin abundance by preventing aberrant PKCθ activation. On the other hand, shRNA-mediated reduction of sortilin in intact C2C12 myotubes inhibited insulin-induced GLUT4 recycling without dampening Akt phosphorylation. We found that the peroxisome proliferator-activated receptor γ agonist troglitazone prevented the palmitate-induced sortilin reduction and also ameliorated insulin-responsive GLUT4 recycling without altering the palmitate-evoked insults on signaling cascades; neither highly phosphorylated PKCθ states nor impaired insulin-responsive Akt phosphorylation was affected. Taken together, our data provide novel insights into the pathogenesis of PKCθ-dependent insulin resistance with respect to insulin-responsive GLUT4 translocation, which could occur not only through defects of insulin signaling but also via a reduction of sortilin, which directly controls trafficking/sorting of GLUT4 in skeletal muscle cells. In addition, our data suggest the insulin-sensitizing action of peroxisome proliferator-activated receptor γ agonists to be at least partially mediated through the restoration of proper GLUT4 trafficking/sorting events governed by sortilin.     

Lekking in Neotropical Owl Butterflies, Caligo illioneus and C. oileus (Lepidoptera: Brassolinae)

We demonstrate that the mating patterns of owl butterflies Caligo illioneus (Cramer)and C. oileus (Felder) are leks. During 1993–1994, we recorded distributions of male and female butterflies and larval hostplants in a lowland Neotropical rain forest in Panama. Caligo illioneus males aggregated along forest edges and defended territories against both conspecifics and males of the related species C. oileus, which exhibited similar behaviors. Male perch sites were not associated with hostplant dispersion or the local abundance of females. However, unmated female C. illioneus were observed to arrive and copulate with males on territories that were located near where streams intersected the roadway. We found some evidence that these leks overlap to form multiple-species aggregations. Caligo illioneus and C. oileus used the same sites at similar frequencies during 1993, a pattern that was repeated during 1994. We could not detect if members of different species were being attracted by similar environmental features or if they were effectively attracting one another to the display sites. Independent of population growth, the abundance of males at a particular site was correlated with the abundance of heterospecific males during 1993, but this pattern was not confirmed in 1994. Overlap in the leks serves as evidence against a resource-based hot-spot hypothesis of lek formation.