Glucocorticoid-stimulated, transcription-independent release of annexin A1 by cochlear Hensen cells.

BACKGROUND AND PURPOSE: The current clinical strategy to protect the auditory organ against inflammatory damage by migrating leukocytes is the local delivery of glucocorticoids. However, the mechanism by which glucocorticoids confer this protection remains unknown. Therefore, we investigated the cellular and molecular targets of glucocorticoids in the cochlea that could be involved in preventing leukocyte migration. EXPERIMENTAL APPROACH: We used microscopy as well as immunocytochemical and microfluidic techniques to elucidate the effect of dexamethasone, hydrocortisone and prednisolone on the cellular and intracellular distribution of annexin A1 (ANXA1) - a glucocorticoid target known to inhibit leukocyte migration by receptor-mediated signalling - in the cochlea and isolated cochlear cells of guinea pigs. KEY RESULTS: All the cells lining the scala media - the cochlear compartment containing the auditory organ - express ANXA1 and the ANXA1 receptor FPR2/ALX is present in the scala media, as well as in other cochlear ducts. The majority of ANXA1 in the scala media is stored inside lipid droplets within cochlear Hensen cells. Glucocorticoids activate a myosin IIC-mediated mechanism that drives ANXA1 from the lipid droplets to the apical region of the Hensen cells, where ANXA1 is released to the external milieu by a process involving ABC transporters. CONCLUSIONS AND IMPLICATIONS: These findings suggest that ANXA1 could be a major mediator of the anti-inflammatory effects of glucocorticoids in the cochlea and identify new molecular targets for prevention of sudden sensorineural hearing loss.

Application of genomics and proteomics for identification of bacterial gene products as potential vaccine candidates.

The ability of bioinformatics to characterize genomic sequences from pathogenic bacteria for prediction of genes that may encode vaccine candidates, e.g. surface localized proteins, has been evaluated. By applying appropriate tools for genomic mining to the published sequence of Haemophilus influenzae Rd genome, it was possible to identify a putative vaccine candidate, the outer membrane lipoprotein, P6. Proteomics complements genomics by offering abilities to rapidly identify the products of predicted genes, e.g. proteins in outer membrane preparations. The ability to identify the P6 protein uniquely from entries in a sequence database from the expected peptide-mass fingerprint of P6 demonstrates the power of proteomics. The application of proteomics for identification of vaccine candidates for another pathogenic bacterium, Helicobacter pylori using two different approaches is described. The first involves rapid identification of a series of monoclonal antibody reactive proteins from N-terminal sequence tags. The other approach involves identification of proteins in outer membrane preparations by 2-D electrophoresis followed by trypsin digestion and peptide mass map analysis. Our combined studies demonstrate that utilization of genome sequences by application of bioinformatics through genomics and proteomics can expedite the vaccine discovery process by rapidly providing a set of potential candidates for further testing.

Mining genomes and mapping proteomes: identification and characterization of protein subunit vaccines.

Currently, there is an extensive and unprecedented effort to obtain the complete nucleotide sequence of the complex genomes of many micro-organisms. In this post-genomic era, based on the availability of the entire genome sequence of an organism, three new disciplines of molecular biology have emerged: genomics, transcriptional profiling and proteomics. All these technologies have the potential to accelerate the process of identifying protective protein antigens as subunit vaccine targets as well as validating and extending the range of available candidate antigens. The progress of these technologies has led to the origination of the science of bioinformatics for management and critical evaluation of the large amount of information generated. Although genomics, transcriptional profiling and proteomics are each based on different principles, there is considerable synergy between them. Appropriate application of any one, or a combination of two or more of these approaches, coupled with bioinformatics, would allow identification of a short-list of vaccine candidates from the entire list of several hundreds to thousands of proteins encoded by the genome. These candidates would then require usual channelling through the subsequent process involving recombinant expression, purification and testing for immunogenicity and protective efficacy.

Evaluation of a 74-kDa transferrin-binding protein from Moraxella (Branhamella) catarrhalis as a vaccine candidate.

An outer membrane protein from Moraxella catarrhalis with a mass of 74-kDa was isolated and evaluated as a vaccine candidate. The 74-kDa protein binds transferrin, and appears to be related to the other proteins from the organism that are reported to bind transferrin. The 74-kDa protein possessed conserved epitopes exposed on the bacterial surface. This is based on the reactivity with whole bacterial cells as well as complement dependent bactericidal activity of sera from mice immunized with the isolated proteins from the O35E and TTA24 isolates. However, there was divergence in the degree of antibody cross-reactivity with the protein from one strain to another. This serotypic divergence was reflected in both the complement-dependent bactericidal activities of the antibodies elicited in mice and the capacity of immune mice to clear the bacteria in a murine pulmonary model. Antibodies affinity purified from human plasma lacked bactericidal activity even though they were reactive with all the tested isolates. The 74-kDa protein appears to be a good vaccine candidate, but more studies are needed to understand its antigenic variability and whether antibodies toward it are protective.

Effect of age on mitogen induced protein tyrosine phosphorylation in human T cell and its subsets: down-regulation of tyrosine phosphorylation of ZAP-70.

Several events of T cell activation have been reported to decline in humans with age. Since protein tyrosine phosphorylation is an early critical event of T cell activation, we performed a systematic analysis of the age-associated changes in the mitogen induced protein tyrosine phosphorylation of human T lymphocytes using SDS-PAGE and Western blotting techniques. Following stimulation with Con A and PHA, an identical pattern of protein tyrosine phosphorylation was observed in the lysates of T cells prepared from seven healthy young adults and eight healthy elderly human subjects. Five different high molecular mass proteins (75, 115, 120, 140 and 170 kDa) were consistently tyrosine phosphorylated in all of the donors from both age groups and peaked between 3 and 10 min. Tyrosine phosphorylation of the above substrates was observed in both CD4 and CD8 subsets. When compared for individual donors from both age groups, variations in the T cell response with regard to net tyrosine phosphorylation for all the substrates was observed. However, the mitogen induced level of tyrosine phosphorylation of only p75 was found to be significantly lower in unfractionated T cells as well as CD4 and CD8 subsets of older subjects than that of young subjects. Using immunoblotting, p75 was identified as ZAP-70, a member of the syk family of protein tyrosine kinases. Understanding of the biochemical basis of the reduced level of tyrosine phosphorylation of ZAP-70 will be helpful in delineating the molecular basis of age-associated impairment of T cell activation.

Isolation and characterization of two proteins from Moraxella catarrhalis that bear a common epitope.

The UspA1 and UspA2 proteins of Moraxella catarrhalis are potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350, 000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100 degreesC. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

Purification of an antigenic vaccine protein by selective displacement chromatography.

A recent advance in the state of the art of displacement chromatography has been the development of selective displacement chromatography. In this process, the bioproduct of interest is selectively displaced while impurities with lower retention are eluted in the induced salt gradient and higher retained impurities are desorbed after the breakthrough of the displacer front. In this manuscript, selective displacement chromatography is employed to purify an antigenic vaccine protein (AVP) from an industrial process stream. Displacers were screened and an operating regime plot was employed to establish appropriate conditions for selective displacement. The selective displacement process was successful and resulted in AVP that was equivalent in purity to product obtained at commercial production scale after conventional step gradient chromatography. Methods used to characterize the purified protein include size-exclusion chromatography, SDS-PAGE, isoelectric focusing, N-terminal amino acid sequence analysis, and amino acid composition analysis. This is the first report of the purification of a commercially and pharmaceutically significant protein using selective displacement chromatography and thereby sets the stage for the implementation of selective displacement chromatography for the downstream processing of biologicals.

Purification and characterization of a phagocytosis inducing factor: role in cell-substratum adherence.

The presence of two different signaling molecules is essential to induce opsonin-independent phagocytosis of particulate activators of the human alternative complement pathway by human monocytes. In addition to the involvement of a low M(r) peptide cytokine or phagocytosis inducing factor (PIF), we have now established that the participation of bacterial lipopolysaccharide is also required. PIF has been demonstrated to be present in human cell lines of different origins, e.g., WISH cells, Raji cells, U937 cells, HL-60 cells and M21 cells. PIF has been purified to apparent homogeneity from the U937 cell line by anion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sephadex G-50. On the basis of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass has been estimated to be approximately 1,600 Da. PIF also plays an important role in the regulation of cell-substratum adherence.

In vivo molecular analysis of cytokines in a murine model of ocular onchocerciasis. I. Up-regulation of IL-4 and IL-5 mRNAs and not IL-2 and IFN gamma mRNAs in the cornea due to experimental interstitial keratitis.

Sclerosing keratitis is the major cause of blindness due to onchocerciasis which results from chronic infection with the filarial parasite Onchocerca volvulus. Using a murine model of onchocercal sclerosing keratitis, we have demonstrated previously that predominantly (> 85%) CD3 + /CD4+ T-cells as well as the IL-2 receptor bearing cells infiltrate into the cornea in vivo during development and progress of the disease. The identification of CD4+ subsets TH1 and TH2 based on the cytokine secretion patterns of murine T-lymphocytes has been useful for understanding the immune basis of resistance and pathogenesis in murine models of several parasitic diseases. The present investigation was carried out to demonstrate whether the local immune response at the corneal lesion due to onchocercal interstitial keratitis correlated with such distinct patterns of cytokine production. For that purpose, mRNA was extracted separately from corneas obtained from the diseased eyes and the normal eyes of A/J mice with onchocercal interstitial keratitis, reverse transcribed and amplified by the polymerase chain reaction with four different cytokine specific primers. In corneas obtained from the eyes affected with onchocercal interstitial keratitis, mRNAs coding for IL-4 and IL-5 were up-regulated compared to the normal eyes having no lesions from the same animals. However, the levels of mRNAs for IL-2 and IFN gamma were found to be the same in the diseased and normal eyes. Taken together, these data suggest that IL-4 and IL-5 producing TH2-lymphocytes are active at the corneal lesion due to onchocercal interstitial keratitis.

Characterization of native pathogenic antigens of Onchocerca volvulus: identification of high molecular mass protein antigens eliciting interstitial keratitis in a guinea pig model.

Sclerosing keratitis is the predominant cause of blindness due to onchocerciasis which is a major human parasitic disease caused by the filarial parasite Onchocerca volvulus. In the present investigation, native pathogenic antigens of O. volvulus which are particularly potent in causing interstitial keratitis were characterized utilizing a guinea pig model. Following demonstration of the protein nature of these antigens using pronase digestion, the crude O. volvulus antigen extract was subjected to stepwise procedures of protein purification. At each stage of purification, pooled antigen fractions were injected into one cornea of presensitized guinea pigs followed by clinical evaluation of stromal inflammation and vascularization at different intervals of time after intrastromal challenge. Initial purification of the pathogenic antigens was carried out in the following order: molecular sieve chromatography on Bio-gel A-5m. anion exchange chromatography on Mono Q followed by DEAE-Sepharose CL-6B and cation exchange chromatography on Mono S. Two out of six different pools from the Mono S column (pool a eluted unbound at 10 mM-NaCl and pool e eluted between 130 mM and 475 mM-NaCl) were found to be most pathogenic. Further purification of Mono S pool a and pool e separately by gel filtration chromatography using Superose 12 demonstrated that the fractions which were most potent in inducing interstitial keratitis contained proteins with approximate molecular masses between 100 and 200 kDa. These results show that minor subfractions of total crude antigens of O. volvulus are largely responsible for induction of experimental interstitial keratitis. We have demonstrated the presence of these antigens in O. volvulus microfilariae by their cross-reactivities with anti-microfilarial antibodies, and hence the relevance of the purified antigens to ocular onchocerciasis in man since sclerosing keratitis is associated with invasion of the cornea by O. volvulus microfilariae. Isolation of these two pathogenic antigen pools represents the practical limits of purification and subsequent animal experiments possible with the available amounts of native parasite material obtained from infected human individuals in the absence of a suitable non-human host or of an in vitro culture system for O. volvulus.

Infiltration of CD4+ T cells into cornea during development of Onchocerca volvulus-induced experimental sclerosing keratitis in mice.

Sclerosing keratitis is the major cause of blindness due to onchocerciasis caused by the parasite Onchocerca volvulus. Although the importance of T cells in the pathogenesis of onchocerciasis has been suggested, their precise role in onchocercal sclerosing keratitis has not yet been defined. Using immunohistological techniques and a murine model of onchocercal sclerosing keratitis, we have performed a temporal analysis of the inflammatory T cells infiltrating into the cornea at Days 4, 7, and 21 following intrastromal challenge with soluble O. volvulus antigens into presensitized mice. The maximum number of CD3+ T cells were observed in the corneal stroma at Day 21 when sclerosing keratitis was most severe. The majority (> 85%) of the CD3+ T cells were CD4+ at all time points. A few infiltrating cells bore IL-2 receptors indicating possible activation of a small fraction of the T cells. These results suggest that CD4+ T cells play an important role in onchocercal sclerosing keratitis.

Role of de novo protein synthesis by human mononuclear leukocytes in opsonin-independent phagocytosis.

Human peripheral blood lymphocytes or the soluble (105,000g) supernatant of the lymphocyte lysate can increase the percentage of human monocytes ingesting particulate activators of the human alternative complement pathway, e.g., rabbit erythrocytes (Chakravarti et al., J. Immunol. 137, 880, 1986). We now show that preincubation of the lymphocytes led to an increase in their augmenting ability. However, no such increase in the augmenting ability of the lymphocytes or soluble supernatant made from these lymphocytes was observed when they were preincubated and added to monocytes in the presence of cycloheximide; rather there was a significant reduction in the ingesting ability of the monocytes. Our results demonstrate that in the case of intact lymphocytes, the observed inhibition was because of (i) inhibition of de novo synthesis of protein(s) in the monocytes during their adherence and (ii) inhibition of de novo synthesis of protein(s) in the lymphocytes, possibly of a signaling molecule necessary for release of the peptide cytokine, phagocytosis-inducing factor (PIF), from the lymphocytes. However, the inhibition observed with soluble supernatant made from lymphocytes preincubated in the presence of cycloheximide was because of the above phenomenon (i) only and indicated that there was no de novo synthesis of PIF during preincubation of the lymphocytes.

Immune-mediated Onchocerca volvulus sclerosing keratitis in the mouse.

Sclerosing keratitis is the major cause of blindness due to onchocerciasis; its pathogenesis is poorly understood. We have previously reported an immune-mediated model of experimental interstitial keratitis in guinea pigs following intrastromal challenge with soluble antigens from Onchocerca volvulus. This model system is ideal for evaluation of pathogenicity of multiple purified antigen preparations; however, reagents necessary for detailed immunologic analysis of the inflammatory cellular infiltrate are not yet available for guinea pigs. Because of the ready availability of these reagents for mice, a mouse model has been developed. The inflammatory response observed in this model was analogous to that seen in human onchocercal sclerosing keratitis as well as in the guinea pig model of onchocercal sclerosing keratitis. Granulocytes were present in the acute inflammatory response, whereas the chronic response showed lymphocytes, plasma cells, and histiocytes. Neovascularization and scarring of the corneal stroma was also observed. This model will be helpful in examining the mechanisms of immunopathogenesis and the contribution of the host genetic background to the disease.

Structural homology of complement protein C6 with other channel-forming proteins of complement.

The amino acid sequence of the amino-terminal half of the complement protein C6 has been found to show overall structural homology with the homologous regions of the channel-forming proteins C7, C8 alpha, C8 beta, and C9. In addition, two specific cysteine-rich segments common to the amino-terminal regions of C7, C8 alpha, C8 beta, and C9 also occur in their expected positions in C6, suggesting functional significance. Two cDNA clones encoding C6 were isolated from a human liver library in the bacteriophage vector lambda gt11. The predicted protein sequence contains an apparent initiation methionine and a putative signal peptide of 21 residues, as well as a site for N-glycosylation at residue 303. The sequence of the C6 protein reported here has 47-52% similarity with C7, C8 alpha, C8 beta, and C9, as well as 31-38% similarity with thrombospondin, thrombomodulin, and low density lipoprotein receptor. The sequence data have been interpreted by using computer algorithms for estimation of average hydrophobicity and secondary structure.

Biochemical characterization of the human complement protein C6. Association with alpha-thrombin-like enzyme and absence of serine protease activity in cytolytically active C6.

Complement protein C6 has been proposed by others to be a serine protease whose activity is obligatory for complement-directed cell lysis. We separated the serine protease (Mr approximately 30,000) activity found associated with apparently homogeneous preparations of C6 from the hemolytically active C6 protein. The protease was characterized as thrombin-like based on substrate specificity, inhibitor profile, and kinetic studies. Although the proteolytic activity of C6 preparations was inhibitable by several inhibitors of serine proteases, the C6 hemolytic activity remained unaffected. Acid-induced (C(5,6)a complex formation between C5 and C6 (protease-free) was demonstrated by ion-exchange fast protein liquid chromatography, reversed-phase high performance liquid chromatography, and reactive cytolytic activity in the presence of C7, C8, and C9; but no cleavage of the alpha-chain of C5 was observed. Diisopropylphosphorofluoridate pretreatment of the components did not affect their ability to generate functionally active (C(5,6)a. Evidently, C6-associated thrombin is not required for formation of functional C(5,6)a. Thus, C6 does not function in the membrane attack pathway of complement as a serine protease. A method for the isolation of homogeneous C6 in the hemolytically fully active form is described. No free sulfhydryl group was detected in C6. The amino acid sequence of 20 amino-terminal residues was determined.

The structure of human complement component C7 and the C5b-7 complex.

The molecular architecture of human complement component C7 was elucidated at several structural levels. The complete primary structure of C7 was derived from the cDNA sequence of clones isolated from a human liver library. C7 is a mosaic protein that consists of 821 amino acids. The amino-terminal two-thirds of C7 has 23-30% homology with complement components C8 and C9. In addition, the carboxyl-terminal third contains four cysteine-rich segments that have overlapping internal homology. The protein is a single polypeptide chain with 28 disulfide bonds and is glycosylated at two sites. Virtually all the cysteines are found in small units of 35-77 amino acids that exhibit homology with those of various proteins including the low density lipoprotein receptor, epidermal growth factor precursor, thrombospondin, and blood coagulation factors IX and X. The secondary structural analysis, estimated by circular dichroism, suggested a high content of beta-sheet (38%) and beta-turns (24%). The tertiary structure, visualized by transmission electron microscopy, indicated a flexible elongated molecule with dimensions of 151 X 59 X 43 A. The quaternary structure of the C5b-7 complex bound to lipid vesicles was observed to be in the form of monomers or dimers. The monomer C5b-7 consists of a leaflet and a long flexible stalk, and the dimer has two leaflets linked through a supercoiled stalk. Membrane binding is mediated by the stalk part of the complexes. Using a radioiodinated photoreactive cross-linking reagent bound to the polar head group of phosphatidylethanolamine, the stalk part of the C5b-7 complex could be labeled preferentially, and it was found to consist mainly of C6 and C7. Thus, C7 plays a major role in bringing about the hydrophilic-amphiphilic transition during the formation of the membrane attack complex, and it serves as a membrane anchor for the C5b-7 complex.

The chemical structure of the C4d fragment of the human complement component C4.

The complete amino acid sequence of the C4d fragment (380 residues long) of the human complement component C4 is presented. Most of the sequence was determined by analysis of CNBr peptides and tryptic peptides obtained from S-carboxymethylated protein. The sequence of the amino terminal 88 residues [Campbell R. D., Gagnon J. and Porter R. R. (1981) Biochem. J. 199, 359-370] and a 106 residue polymorphic segment of C4d [Chakravarti D. N., Campbell R. D. and Gagon J. (1983) FEBS Lett. 154, 387-390] was extended. Some overlaps not provided by the protein sequence analysis were obtained from the amino acid sequence predicted by the nucleotide sequence [Belt K. T., Carroll M. C. and Porter R. R. (1984) Cell 36, 907-914]. The present protein sequence data provide information for the isolation of all the CNBr and succinylated tryptic peptides of C4d. In addition to the polymorphism previously described, two other sets of polymorphic amino acid residues at positions 153 (Ile/Ser) and 154 (Gln/Ala) have been identified. The major site of glycosylation has been shown to be an asparagine residue located in the sequence -Asn-Val-Thr- in the carboxy terminal end of C4d. A remarkable difference in the predicted secondary structure of C4d arising from one set of four polymorphic residues in a stretch of six residues and another single polymorphic residue suggests a structural basis for the origin of the different chemical reactivities of the C4 isotypes (C4A and C4B) and their serological difference in the expression of Rodgers or Chido blood group antigens. Possible non-covalent membrane attachment sites have been suggested from the hydropathy profile. Comparison of the C4d sequence with human C3, C5 and alpha 2-macroglobulin revealed extended stretches of sequence similarity (between 19 and 38% homology) with the corresponding regions of these proteins.

Amino acid sequence of a polymorphic segment from fragment C4d of human complement component C4.

The amino acid sequence of a segment of 106 residues of C4d has been determined by automated sequence analysis of fragments obtained by CNBr cleavage and enzymic digestion with trypsin. Polymorphism has been detected at 3 positions. Residues 9 and 12 are either valine and leucine or alanine and arginine, respectively. Residue 102 is either valine or arginine. When comparing the protein sequence with the nucleic, acid sequence [Carroll, M. and Porter, R.R. (1983) Proc. Natl. Acad. Sci. USA 80, in press] alanine or serine are found at position 98. These results may in part help to explain the inherited variants of human C4 seen on gel electrophoresis.