Eosinophil cationic protein damages protoscoleces in vitro and is present in the hydatid cyst.

Eosinophils are locally recruited during the establishment and chronic phases of cystic hydatidosis. This study provides evidence that eosinophil cationic protein (ECP), one of the major components of eosinophil granules, can damage Echinococcus granulosus protoscoleces (PSC). The toxicity of ECP was investigated in vitro by following parasite viability in the presence of this protein. ECP was found to damage PSC at micromolar concentrations; the effect was blocked by specific antibodies and heparin, and was more severe than the one caused by similar concentrations of RNase A, suggesting that the cationic nature of ECP, and not its ribonuclease activity, is involved in toxicity. This observation may highlight the capacity of eosinophils to control secondary hydatidosis, derived from PSC leakage from a primary cyst. To further assess the relevance of the previous result during infection, the presence of eosinophil proteins was investigated in human hydatid cysts. ECP was found to be strongly associated with the laminated layer of the cyst wall, and present at micromolar concentrations in the hydatid fluid. Overall, these results demonstrate that eosinophils degranulate in vivo at the host-parasite interface, and that the released ECP reaches concentrations that could be harmful for the parasite.

VCAM-1 is more effective than MAdCAM-1 in supporting eosinophil rolling under conditions of shear flow.

The ability of the alpha4 integrin counterligands vascular cell adhesion molecule (VCAM)-1 or mucosal addressin (MAd)CAM-1 to support eosinophil rolling or firm adhesion under conditions of physiologic flow has not been delineated. Using a parallel plate flow chamber in vitro and intravital microscopy in vivo, we demonstrate that eosinophil rolling and adhesion on VCAM-1 is mediated by both alpha4beta1 and alpha4beta7 integrins. Eosinophils rolled equally efficiently on both VCAM-1 2 domain and VCAM-1 7 domain, suggesting that the N-terminal 2 domains of VCAM-1 are sufficient to support eosinophil rolling under conditions of flow. Furthermore, activation of the eosinophil beta1 integrin with monoclonal antibody (mAb) 8A2 resulted in both resistance to shear stress-induced detachment from VCAM-1 in vitro and in stable arrest of rolling eosinophils on interleukin (IL)-1beta-stimulated venules in vivo. Eosinophils rolled less efficiently on MAdCAM-1- than on VCAM-1-coated coverslips under conditions of flow. However, eosinophils firmly adhered as efficiently to MAdCAM-1 as to VCAM-1. Overall, these results demonstrate that both VCAM-1 and MAdCAM-1 can support eosinophil firm adhesion under conditions of flow. In contrast, VCAM-1 is significantly more efficient than MAdCAM-1 in supporting eosinophil rolling under conditions of flow. (Blood. 2000;95:592-601)

Function of the factor I modules (FIMS) of human complement component C6.

In order to elucidate the function of complement component C6, truncated C6 molecules were expressed recombinantly. These were either deleted of the factor I modules (FIMs) (C6des-748-913) or both complement control protein (CCP) modules and FIMs (C6des-611-913). C6des-748-913 exhibited approximately 60-70% of the hemolytic activity of full-length C6 when assayed for Alternative Pathway activity, but when measured for the Classical Pathway, C6des-748-914 was only 4-6% as effective as C6. The activity difference between C6 and C6des-748-913 for the two complement pathways can be explained by a greater stability of newly formed metastable C5b* when produced by the Alternative Pathway compared with that made by the Classical Pathway. The half-lives of metastable C5b* and the decay of (125)I-C5b measured from cells used to activate the Alternative Pathway were found to be about 5-12-fold longer than those same parameters derived from cells that had activated the Classical Pathway. (125)I-C5 binds reversibly to C6 in an ionic strength-dependent fashion, but (125)I-C5 binds only weakly to C6des-FIMs and not at all to C6des-CCP/FIMs. Therefore, although the FIMs are not required absolutely for C6 activity, these modules promote interaction of C6 with C5 enabling a more efficient bimolecular coupling ultimately leading to the formation of the C5b-6 complex.

The architectural transition of human complement component C9 to poly(C9).

Several regions of C9 including three cysteine-rich modules homologous to those in thrombospondin (TS), the low density lipoprotein receptor (LDL), the epidermal growth factors (EDGF), as well as two middle sections of the polypeptide chain were expressed in bacteria. Antibodies derived from these segments were used to probe the relative exposure of epitopes in C9 and poly(C9) using ELISAs. The results indicated that the TS and LDL modules are fully exposed in both monomer and polymer; however, the middle region of the polypeptide chain is buried in the monomer but external in the polymer. Using specified conditions, Fab fragments to the TS and LDL modules did not block C9 polymerization, but those to the middle region of the polypeptide chain and to some extent to the EDGF module did so. Immuno-electron microscopy of poly(C9) indicated that the C9 polypeptide chain assumes a 'U' shape, in which the TS and LDL modules are located on the upper rim. The EDGF module is located on the lower edge of the upper rim, and midsection of the polypeptide chain constructs the barrel of the tubule. Computer assisted contrast enhancement of select electron micrograph images of poly(C9) allowed the clear visualization of each subunit. These were seen to have a volute shape. The upper rim is composed of whorls that are apparently not in lateral contact. It is concluded that the TS and LDL modules do not participate directly in polymerization but cover the hydrophobic central region of the polypeptide chain in the monomer. As a consequence of circular polymerization the midsection of the polypeptide chain becomes exposed as each C9 lengths to fashion a volute form. reserved.

Deposition of complement C3 and factor H in tissue traumatized by burn injury.

Activation of complement is known to accompany burn injury. To study deposition of complement proteins within tissue traumatized by burn we employed the technique of intravital microscopy using a murine dorsal skinfold chamber model. C3, factor H, factor B, HSA, and transferrin were labeled fluorescently and injected into the tail vein of mice which had been subjected to a small third degree burn within the skin fold. Only C3 and factor H deposited within blood vessels of the traumatized tissue. Binding was specific because it occurred only in and proximal to burn sites, and neither C3 nor factor H was observed to accumulate in blood vessels of healthy tissue. Furthermore, fluorescently labelled HSA, factor B, and transferrin all failed to deposit at or around burn loci. The deposition of C3 and factor H occurred within 10 min of injury and was intravascular occurring in major blood vessels, capillaries, and post-capillary venules, with little evidence of accumulation in the interstitium. Since both C3 fragments and factor H are recognized as adhesion molecules by granulocyte receptors, these deposited proteins could promote leukocyte accumulation, thereby contributing to an initiation of an inflammatory cascade at a site of burn injury.

A comparison of C3a and C5a-mediated stable adhesion of rolling eosinophils in postcapillary venules and transendothelial migration in vitro and in vivo.

The comparative ability of the complement anaphylatoxins C3a and C5a to mediate leukocyte adhesion and transendothelial migration in vivo and in vitro was investigated. Superfusion of IL-1beta-stimulated rabbit mesentery with C3a resulted in a rapid and stable adhesion of rolling eosinophils, but not neutrophils, to postcapillary venules. However, C3a failed to evoke subsequent transmigration of the adherent eosinophils. In contrast, C5a induced both the rapid activation-dependent firm adhesion and transmigration of eosinophils and neutrophils through venular endothelium. C3a induced selective shedding of L-selectin and an increase in alphaMbeta2 integrin expression on eosinophils but not neutrophils, while C5a induced shedding of L-selectin and up-regulation of alphaMbeta2 integrin on both eosinophils and neutrophils. Both C3a- and C5a-dependent adhesion to venular endothelium was blocked by ex vivo treatment of eosinophils with anti-alpha4 and anti-beta2 integrin mAbs. In vitro, both C3a (but not C3a(desArg)) and C5a (including C5a(desArg))-dependent transmigration of eosinophils across IL-1beta-stimulated endothelial monolayer was mediated by alpha4beta1 and alphaMbeta2 integrins. Overall these studies suggest that C3a is eosinophil-specific chemotactic mediator that influences selectively eosinophil adhesion but not transmigration in vivo. C5a in contrast is a complete activator of integrin-dependent adhesion as well as transmigration of eosinophils and neutrophils.

Crystallization of human complement component C5.

Human complement component C5 has been crystallized using a low-salt batch technique. The crystals are large hexagonal bi-pyramids often larger than 1.5 mm. Although these crystals were grown in low salt (0.1 M NaCl), they are remarkably stable for at least 2 months at 281 K and they are not dissolved in aqueous buffers containing up to 2 M sodium chloride. The space group is P3121 or P3221, and the cell parameters were determined to be a = 144.9, b = 144.9, c = 243.1 A; alpha = 90 degrees, beta = 90, gamma = 120 degrees. At room temperature and cryo-temperatures the crystals diffract at best to 6 A using rotating-anode X-ray sources. Using synchrotron radiation with cryoprotection using 40%(v/v) PEG 400 the resolution limit can be extended to 3.3 A. In both cases the crystals show significant anisotropy, with relatively weaker reflections at higher resolution in the a*b* plane.

Human polymorphonuclear leukocytes adhere to complement factor H through an interaction that involves alphaMbeta2 (CD11b/CD18).

The work presented here demonstrates that human complement factor H is an adhesion ligand for human neutrophils but not for eosinophils. The adherence of polymorphonuclear leukocytes (PMNs) to plastic wells coated with factor H depended on divalent metal ions and was augmented by C5a and TNF-alpha. PMN adhesion to factor H in the presence or absence of C5a was blocked specifically by mAbs against CD11b or CD18. Affinity purification using factor H Sepharose followed by immunoprecipitation using mAbs to various integrin chains identified Mac-1 (CD11b/CD18) as a factor H binding receptor. The presence of surface bound factor H enhanced neutrophil activation resulting in a two- to fivefold increase in the generation of hydrogen peroxide by PMNs stimulated by C5a or TNF-alpha. When factor H was mixed with PMNs, 1.4 to 3.8-fold more cells adhered to immobilized heparin or chondroitin A. In addition, augmented adhesion of PMNs was measured when factor H, but not HSA or C9, was absorbed to wells that were first coated with heparin or chondroitin A. The adhesion of PMNs to glycosaminoglycan-factor H was blocked by mAbs to CD11b and CD18. These studies demonstrate that factor H is an adhesion molecule for human neutrophils and suggest that the interaction of factor H with glycosaminoglycans may facilitate the tethering of this protein in tissues allowing factor H to serve as a neutrophil adhesion ligand in vivo.

Elucidation of the disulfide-bonding pattern in the factor I modules of the sixth component (C6) of human complement.

Complement component C6 is known to contain two factor I modules in tandem at its C-terminus. To localize the disulfide bridges in those domains, native C6 was cleaved with trypsin, followed by subtilisin. The resulting digests were separated by reversed-phase HPLC, and all of the potential cystine-containing fragments were detected by a fluorescence assay and amino acid composition analyses. Final identification of the disulfide bonds was achieved by Edman degradation of the corresponding peptides. From the data gained a 1-3, 2-9, 4-7, 5-10, 6-8 pattern was determined (Cys752-Cys802, Cys763-Cys780, Cys765-Cys816, Cys772-Cys795, Cys841-Cys852, Cys846-Cys898, Cys859-Cys876, Cys861-Cys911, Cys867-Cys891). These findings are compared with the strongly related complement components C7 and factor I.

Characterization of complement C6 deficiency in a PVG/c rat strain.

Complement C6 plays an important role in the effector phase of complement-mediated cell lysis. Recently, a PVG/c rat strain deficient in haemolytic C6 activity was discovered. In the present study we show that these rats lack both antigenic and functional C6, and that repetitive immunization of these rats with PVG/c+ serum results in generation of specific anti-rat C6 antibodies. The observed absence of rat C6 was further investigated at the genomic and transcriptional level using a 492-bp cDNA of rat C6, cloned from a rat liver cDNA library using full length human C6 as a probe. Northern blot analysis revealed the presence of C6 mRNA in livers of both PVG/c- and PVG/c+ rats, corresponding to a size of approximately 3.3 kb, although the level of C6 mRNA expression was approximately 100-fold less in PVG/c- rats. In addition, using rat C6-specific primers, positive signals were obtained in kidneys of both rat strains by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Southern blot analysis of digested genomic DNA did not reveal evidence for large C6 gene deletions. We conclude that the lack of C6 protein in the PVG/c- rat strain is not due to a (large) C6 gene deletion, but presumably is caused by an unstable mRNA or a point mutation in the C6 gene resulting in an aberrant transcription of the C6 gene. Alternatively, a gene coding for a product involved in C6 biosynthesis that acts in trans may carry a mutation.

Chemotactic factors released in culture by intact developing and healing skin lesions produced in rabbits by the irritant sulfur mustard.

Development, peak and healing lesions were induced in the skin of rabbits by topical applications (on different days) of the chemical irritant sulfur mustard (SM). Immediately after the rabbits were euthanized, the intact lesions were excised and organ-cultured for 17 to 20 hours. The culture fluids from early, peak and healing SM lesions all showed high chemotactic activity for both PMN and MN. This finding suggests that the PMN and MN, seen microscopically in tissue sections of the lesions, were entering continuously, even during the healing process. The chemotaxins identified were the eicosanoid LTB4, the chemokine IL-8, and proteases producing the complement fragment C5a. Other studies from our laboratory showed that the number of cells containing IL-1, IL-8, MCP-1, and GRO mRNAs was increased in SM lesions. Chemotactic activity was released by both live and dead (frozen and thawed) cell suspensions of PMN, MN, and fibroblasts, suggesting that these cells were major sources of the chemotaxins produced by the SM lesion explants. Explants of normal skin produced considerable chemotactic activity for MN, but not for PMN. Chemotactic activity for PMN, and the release of LTB4, IL-8 and proteases cleaving C5 to C5a, occurred only in explants infiltrated by leukocytes.

E-selectin preferentially supports neutrophil but not eosinophil rolling under conditions of flow in vitro and in vivo.

The selectin family of adhesion molecules is composed of the L-, E-, and P-selectins, which promote leukocyte rolling during inflammation. Although E-selectin supports neutrophil and lymphocyte rolling, its ability to mediate eosinophil rolling under conditions of flow in vitro and in vivo has not been determined. Using function-blocking mAbs raised against rabbit E-selectin, we have determined whether E-selectin supports human eosinophil rolling in comparison to human neutrophil rolling in IL-1-stimulated rabbit mesenteric venules utilizing intravital microscopy. Anti-rabbit E-selectin mAbs 8B9 and 8G9 were found to inhibit neutrophil rolling but had no significant effect on eosinophil rolling. Likewise, mAb 8B9 F(ab')2 fragments were found to block neutrophil rolling, but did not significantly alter the flux of rolling eosinophils. Isotype-matched Abs and a nonblocking anti-rabbit E-selectin mAb 2A5 failed to inhibit both neutrophil and eosinophil rolling on venular endothelium. In support of these in vivo observations, significant numbers of human neutrophils but not eosinophils were found to avidly roll on monolayers of E-selectin transfectants under physiologic condition of flow in vitro. Under subphysiologic conditions of shear (0.17-0.5 dyn/cm2), eosinophils rolled on E-selectin, albeit in lower numbers (three- to sevenfold) compared with neutrophils. In addition, the rolling velocity of eosinophils was significantly higher compared with neutrophils on E-selectin transfectants. These studies suggest that at physiologic shear rates, E-selectin is likely to function as a major vascular adhesion receptor in mediating neutrophil but not eosinophil rolling in inflamed postcapillary venules.

Cleavage of human complement component C5 by cysteine proteinases from Porphyromonas (Bacteroides) gingivalis. Prior oxidation of C5 augments proteinase digestion of C5.

Since severe periodontitis is characterized by an acute inflammatory response with cellular infiltration and microbial overgrowth, plasma proteins could be exposed to both proteinases and oxidants released from the granulocytes, as well as to proteinases from the microorganisms. When human complement component C5 was digested by cysteine proteinases (i.e. gingipain-R and gingipain-K) from Porphyromonas gingivalis, limited cleavage of the C5 molecule was observed. If C5 was first oxidized by hydroxyl radicals, these gingipains converted modified C5 to fragments that exhibited significantly greater pro-inflammatory activity than did digests of unmodified C5. After cleavage of oxidized C5 by gingipain-R, the digest exhibited measurably greater neutrophil enzyme release and chemotaxis of human polymorphonuclear leukocytes (PMNs) compared with the activities of unoxidized C5 digests. Gingipain-K generates virtually no polarization or chemotactic activity of human PMNs from C5, nor is enzyme release stimulated by these C5 digests. However, when oxidized C5 was digested by gingipain-K, human PMNs were stimulated for polarization, chemotaxis and enzyme release indicating that an active fragment had been generated. Proteolysis of oxidized C5 evokes greater neutrophil activation than does proteolysis of unoxidized protein, a fact which supports the hypothesis that oxidation and proteolysis may be coupled to enhance the destructive effects of the inflammatory process. These results, in which digests of both oxidized and unmodified complement component C5 were evaluated, support the general concept that oxidation and proteolysis may participate cooperatively in amplifying both the severity and duration of the inflammatory reaction.

The extracellular neutral cysteine proteinase of Entamoeba histolytica degrades anaphylatoxins C3a and C5a.

We have shown previously that the extracellular cysteine proteinase of Entamoeba histolytica trophozoites activates the alternative pathway of complement by specifically cleaving C3. This unique mechanism of complement activation leads to passive lysis of nonpathogenic, but not of pathogenic strains. In an attempt to investigate the relationship between the cleavage of complement components C3 and C5 and the pathogenesis of amebiasis, we investigated the production of the anaphylatoxins C3a and C5a, which have diverse effects on the host immune response. The concentration of proteinase required to cleave purified C5 was at least 5 to 10 times that needed for C3 cleavage, but these levels are easily obtainable as demonstrated by cleavage of 125I-labeled C5 during incubation with purified trophozoites. When the C3a-like cleavage fragments were purified by gel filtration, they were found to be extensively degraded during a 1-h incubation of C3 with the proteinase. Subsequent evaluations of the C3a- and C5a-like cleavage products generated earlier in the reaction using immunoblots and cellulose acetate electrophoresis revealed rapid degradation, even during incubation periods as short as 5 min. Because C-terminal fragments as small as 20 amino acid residues can mimic the biologic functions of C3a or C5a, we tested cleavage products for activity. In sensitive bioassays, including guinea pig platelet aggregation for C3a activity and chemotaxis for C5a activity, we demonstrated that proteolysis renders these molecules inactive. These studies suggest that the extracellular cysteine proteinase of E. histolytica, which is capable of activating the complement system, may also provide a mechanism to circumvent normal host immunity by inactivating the proinflammatory factors C3a and C5a.

Structure of the human C7 gene and comparison with the C6, C8A, C8B, and C9 genes.

The seventh component of complement is a single chain plasma glycoprotein that is involved in the cytolytic phase of complement activation. We have determined the structure of the C7 gene, which is encoded by 18 exons whose sizes vary from 56 to 244 bp. For the most part, the exons do not correspond to the protein homology units. However, two intron/exon boundaries occur at junctions between different functional parts of the protein. The first is at a site between the end of the C9 homology unit and the carboxyl-terminal extension which is also a feature of C6. The second of these boundaries occurs between the regions encoding two pairs of cysteine-rich modules (the short consensus repeats and the factor I modules) located in the carboxyl-terminal part of C7. In contrast to the exons, the introns range considerably in size from 0.5 to 8.5 kbp. The complete analysis indicates that the gene encoding C7 is approximately 80 kbp in length. We show here that the C7 gene is highly homologous to that for C6, and also to C8A, C8B, and C9, confirming and extending the published data. With the exception of exon 1, all intron/exon boundaries are preserved with respect to phase when compared with C6.

The fractionation of human plasma proteins. I. Affinity purification of human complement properdin.

A method was developed for the affinity purification of human complement properdin. The preparation is part of an integrated scheme in which over 20 human plasma proteins can be recovered in a highly purified form. The yield of properdin was 5.9 mg from 3 liters of plasma, amounting to a 28% recovery. The crucial step in the purification was the application of affinity chromatography using C3b-ester-Sepharose.

The fractionation of human plasma proteins. II. The purification of human complement proteins C3, C3u, and C5 by application of affinity chromatography.

We have devised a purification scheme for human complement proteins C3, C3u, and C5. Affinity chromatography is employed to isolate each of these three proteins in a highly purified form. Typical yields were 910, 30, and 53 mg for C3, C3u, and C5, respectively, from 3 liters of human plasma. These yields reflect 31 and 32% recoveries for C3 and C5, respectively. The major advantages of this protocol over other published procedures are the application of affinity chromatography to yield high quality purified complement components, and the fact that the purification of C3, C3u, and C5 is part of a broader scheme designed for the isolation of over 20 plasma proteins from a single batch of plasma.

The fractionation of human plasma proteins. III. Purification of complement factors D and I using affinity chromatography.

A purification protocol for complement factors D and I was designed as part of a larger scheme for the purification of more than 20 human plasma proteins. The key affinity step in the purification of both serine proteases, factors D and I, was affinity chromatography using benzamidine Sepharose. The recoveries were 19 and 12% for factors D and I, respectively.

The size, shape and stability of complement component C9.

Electron microscopy of specimens of C9 tilted through 90 degrees visualized this protein to be a globular ellipsoid with dimensions of 77 x 70 x 52 A. To check the congruence of this observation with physical properties of the molecule, hydrodynamic parameters for C9 were determined. From this work a frictional ratio of 1.32 was calculated. C9 was compared with several other proteins of similar frictional ratios whose tertiary structures are known. All examples found of such proteins whose frictional ratios were between 1.26 and 1.37 are either heart-shaped or globular ellipsoids, but none are prolate ellipsoids. By comparison the size and shape of C9 determined by electron microscopy are congruent with its hydrodynamic parameters. Both electron microscopy and physical measurements suggest that the length (110-120 A) of C9 determined by neutron and X-ray scattering experiments is an overestimate. The source of the discrepancy was identified by the demonstration that the high concns of C9 employed in neutron and X-ray scattering work lead to aggregation of the protein. Thus, investigations involving neutron and X-ray scattering were measuring polydisperse solutions of C9. The deduced value of the radius of gyration from that work (33-35 A) is now recognized as being statistical and significantly higher than the correct value of monomeric C9 (26 A), which was calculated from electron microscopy measurements. Also high-resolution electron microscopy clearly visualized poly(C9) to be a barrel-stave construct. These results suggest that monomeric C9 must undergo a major conformational alteration to extend by 55-70 A in order to self-associate laterally in order to fashion the cylindrical poly(C9).