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M T Michalek

Publications and source records attributed to M T Michalek.

13 recordsLinked to original sources
PubMed1998

Effects of particulate and soluble (1-3)-beta-glucans on Ca2+ influx in NR8383 alveolar macrophages.

Particulate and soluble (1-3)-beta-glucans are effective in preventing infections by enhancing macrophage and neutrophil functions. However, the mechanisms triggering these enhanced cellular responses are essentially unknown. We recently demonstrated that zymosan, a particulate (1-3)-beta-glucan receptor agonist, caused an influx of Ca2+ in NR8383 rat alveolar macrophages (AMs) and a resulting increase in intracellular Ca2+ (Zhang et al., J. Leukoc. Biol. 62 (1997) 341-348). Since Ca2+ is important in mediating leukocyte responses, we investigated whether other (1-3)-beta-glucans also alter Ca2+ mobilization in AMs. Particulate and soluble (1-3)-beta-glucans derived from Saccharomyces cerevisiae were used in these studies. Like zymosan, particulate (1-3)-beta-glucan (WGPs) caused a concentration-dependent increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+ and by SKF96365, an inhibitor of receptor-operated Ca2+ channels. When three different soluble (1-3)-beta-glucans, with molecular weights of approximately 11,000, 150,000, and 1,000,000 Da, were tested alone for effects on Ca2+ responses, the low molecular weight (1-3)-beta-glucan produced no effect and the intermediate and high molecular weight (1-3)-beta-glucans caused only a small increase in [Ca2+]i. Interestingly, however, all three soluble (1-3)-beta-glucans could significantly reduce the Ca2+ responses induced by a subsequent exposure to either WGPs or zymosan. These results demonstrate that: 1) particulate (1-3)-beta-glucan activates Ca2+ influx in NR8383 macrophages through receptor-operated Ca2+ channels; 2) soluble (1-3)-beta-glucans do not strongly activate Ca2+ influx in these cells; and 3) soluble (1-3)-beta-glucans significantly inhibit Ca2+ influx induced by WGPs or zymosan. Soluble (1-3)-beta-glucans are likely to prevent Ca2+ influx by competitively binding to the (1-3)-beta-glucan receptors recognizing zymosan and WGPs. The smaller Ca2+ influx induced by soluble (1-3)-beta-glucans may represent only a partial activation of post-receptor signal transduction pathways necessary for inducing Ca2+ influx.

Adjuvants, Immunologic↗
PubMed1996

Chemical denaturation and modification of ovalbumin alters its dependence on ubiquitin conjugation for class I antigen presentation.

Class I presentation of microinjected native OVA by a temperature-sensitive ubiquitin conjugation mutant, ts85, but not wild-type murine cells, was markedly inhibited following incubation at a nonpermissive temperature. In contrast, the nonpermissive temperature did not affect class I presentation of a minimal OVA peptide expressed in the cytosol. Therefore, these results provide a second example in which a temperature sensitive mutation in the ubiquitin conjugation pathway inhibits MHC class I presentation of native OVA. Surprisingly, incubation at the nonpermissive temperature did not inhibit class I presentation of chemically denatured and alkylated OVA microinjected into the cytosol of mutant cells. Similarly, the presentation of endogenously synthesized OVA (which is expressed from a recombinant vaccinia virus and, presumably, is misfolded in the cytosol) was also not inhibited in both mutant cell lines. Methylation of the lysine groups in denatured OVA, which blocks ubiquitin conjugation, reduced but did not eliminate the presentation of denatured OVA, providing evidence for both ubiquitin-dependent and ubiquitin-independent pathways for class I presentation. In contrast, a proteasome inhibitor blocked class I presentation of all forms of OVA, while a control peptide aldehyde was not inhibitory. These results indicate that modification of the structure of a protein can influence its requirements for ubiquitin conjugation for efficient class I presentation, with the key alteration possibly being the loss of proper conformation. However, regardless of the form of the Ag, the proteasome appears to be required for generating peptides from both endogenously synthesized and microinjected OVA for class I presentation.

Amino Acid Sequence↗
PubMed1995

Rate of antigen degradation by the ubiquitin-proteasome pathway influences MHC class I presentation.

The effect on MHC class I Ag presentation of enhancing a protein's rate of degradation by the ubiquitin-proteasome pathway was investigated. In extracts of mouse B-lymphoblasts and reticulocytes, as in rabbit reticulocytes, proteins with acidic or basic N-termini are conjugated to ubiquitin and degraded by the 26S proteasome very rapidly. We found that the rate of MHC class I presentation of microinjected beta-galactosidase was enhanced when this antigenic protein was modified with such a destabilizing amino-terminal residue. This enhanced presentation was inhibited by blocking potential ubiquitination sites on the protein through methylation of amino groups and by peptide aldehyde inhibitors of the proteasome. Furthermore, in B lymphoblast cell extracts, the rapid degradation of these beta-galactosidase constructs required ATP and ubiquitin and was blocked by inhibitors of proteasomes. Their rates of degradation in extracts correlated with their rates of class I Ag presentation in vivo. These results indicate that ubiquitin conjugation is a key rate-limiting step in Ag presentation and provide further evidence for a critical role of ubiquitin and the 26S proteasome in generating MHC class I-presented peptides.

Amino Acid Sequence↗
PubMed1993

A role for the ubiquitin-dependent proteolytic pathway in MHC class I-restricted antigen presentation.

The degradation of most cellular proteins starts with their covalent conjugation with ubiquitin. This labels the proteins for rapid hydrolysis to oligopeptides by a (26S) proteolytic complex containing a (20S) degradative particle called the proteasome. Some system in the cytosol also generates antigenic peptides from endogenously synthesized cellular and viral proteins. These peptides bind to newly synthesized class I major histocompatibility complex molecules in the endoplasmic reticulum and peptide/class I complexes are then transported to the cell surface for presentation to cytotoxic T cells. How these peptides are produced is unknown, although a modification that promotes ubiquitin-dependent degradation of a viral protein enhances its presentation with class I13 and indirect evidence suggests a role for proteolytic particles closely resembling and perhaps identical to the proteasome. Using cells that exhibit a temperature-sensitive defect in ubiquitin conjugation, we report here that non-permissive temperature inhibited class I-restricted presentation of ovalbumin introduced into the cytosol, but did not affect presentation of an ovalbumin peptide synthesized from a minigene. These results implicate the ubiquitin-dependent proteolytic pathway in the production of antigenic peptides.

Amino Acid Sequence↗
PubMed1992

The class II MHC-restricted presentation of endogenously synthesized ovalbumin displays clonal variation, requires endosomal/lysosomal processing, and is up-regulated by heat shock.

LB27.4 cells (a B lymphoblastoid APC) were transfected with a plasmid containing an OVA cDNA. Functional analysis of six independent clones yielded three patterns of MHC-restricted presentation of the endogenously synthesized OVA. A clone displayed either: 1) strong class I and class II-restricted presentation, 2) strong class I but little or no class II-restricted presentation or, 3) only a modest class I-restricted presentation. There was no clonal variation in class II-restricted presentation of exogenous Ag or in the amount of surface class I or II molecules. Heat shock increased the presentation of endogenous but not exogenous Ag with class II. These results indicate that an endogenously synthesized Ag both constitutively and during heat shock can gain access to the class II, MHC-restricted, presentation pathway. The amount of OVA synthesized by a cell correlated with whether OVA-class II complexes were detected. However, the amount of OVA secreted into the extracellular fluid was not sufficient to sensitize APC, which suggests that endogenously synthesized OVA enters the class II pathway of Ag presentation by an intracellular route rather than by an extracellular/reuptake route. Also, the functional and quantitative analysis of the clones suggests that endogenously synthesized OVA was presented more efficiently with class I as compared to class II-MHC molecules. Leupeptin and chloroquine inhibited the class II-restricted presentation of endogenously synthesized OVA. Together these results indicate that endogenously synthesized OVA can gain access to an endosomal/lysosomal compartment via an intracellular route and be processed and presented in association with class II-MHC molecules.

Animals↗
PubMed1991

Weak base amines can inhibit class I MHC-restricted antigen presentation.

This report describes the effects of NH4Cl, CH3NH2, and chloroquine on class I and II MHC-restricted Ag presentation. OVA-specific T-T hybridomas were used to detect processed OVA in association with class I, H-2Kb, and class II, I-Ad/b, molecules on a B lymphoblastoid APC. OVA, internalized by APC under hypertonic conditions, was presented in association with class I and II MHC molecules. Treating the APC with NH4Cl or CH3NH2 inhibited class I- and II-restricted Ag presentation. In contrast, chloroquine markedly inhibited class II, but not class I-restricted Ag presentation. Controls indicated that drug-treated APC were fully competent to interact with T cells and present processing-independent antigenic peptides in association with both class I and II MHC molecules. NH4Cl and CH3NH2 did not inhibit the uptake of radiolabeled Ag by the APC. After the proteolytic removal of H-2Kb from the surface of APC, NH4Cl and CH3NH2-treated and control APC regenerated identical amounts of surface H-2Kb and this regeneration required de novo protein synthesis. These latter results indicate that NH4Cl and CH3NH2 can inhibit Ag presentation without affecting the synthesis, transport, or surface expression of H-2Kb. Also, NH4Cl did not affect the transport of H-2Db to the surface of mutant RMA-S cells that were cultured with exogenous peptides. Taken together these results strongly suggest that NH4Cl and CH3NH2 but not chloroquine can inhibit a critical and early intracellular step in class I-restricted Ag presentation while simultaneously inhibiting class II-restricted Ag presentation.

Amines↗
PubMed1990

Role of ICAM-1 in antigen presentation demonstrated by ICAM-1 defective mutants.

We report a methodology for selecting APC with mutations that have impaired their ability to present Ag to T cells. A20 B lymphoblastoid cells were mutagenized and then repeatedly cocultured with murine T-T hybridomas in the presence of specific Ag. During these cocultures, the T-T hybridomas kill the competent APC, allowing the outgrowth of inactive variants. Two variants, A20.M1 and A20.M2, were isolated and studied in detail. These variants are impaired in their ability to present multiple Ag to T cells. This defect is also observed for the presentation of processing independent peptides by fixed APC indicating that a lesion exists in a post-Ag processing step. The level of expression of MHC molecules is unaffected and the functional defect in the APC is not localized to a particular MHC molecule. In contrast, these mutants were found to have a selective decrease in the expression of the murine homolog of ICAM-1, and the residual ability of these cells to present Ag was not blocked by anti-ICAM-1 mAb. Conversely, Ag presentation by the wild-type A20 is inhibited by anti-ICAM-1 mAb. Similarly, anti-LFA-1 mAb inhibited the response of T cells to Ag presented by the wild-type A20 to a much greater degree than by the mutant cells, indicating that LFA-1 is involved in interaction of T cells with the former, but not latter, APC. In the apparent absence of a contribution of LFA-1 to the T cell-APC interaction, either as a result of mAb blocking or the disruption of the APC membrane, the mutant and wild-type APC have a similar level of Ag-presenting activity. Reconstitution of ICAM-1 expression in these mutants by transfection with murine ICAM-1 cDNA fully restores their ability to present Ag. Together these results demonstrate that a murine ICAM-1 homolog is expressed on A20 B cells, where it functions as a major cell interaction molecule. The degree of functional impairment in these mutant APC gives insight into the contribution of cell interaction molecules to efficient Ag presentation and T cell-B cell interaction. Finally, these results also demonstrate the feasibility of selecting APC with mutations affecting Ag presentation.

Animals↗
PubMed1989

Two genetically identical antigen-presenting cell clones display heterogeneity in antigen processing.

Evidence from various antigen systems suggests that antigen processing can be one factor that determines the repertoire of immunogenic peptides. Thus, processing events may account for some of the disparity between the available and expressed helper T-cell repertoires. In this report, we demonstrate that the immunodominant T-cell determinant in ovalbumin [p323-339; ovalbumin-(323-339) heptadecapeptide] is processed differently by two genetically identical antigen-presenting cell lines, M12 and A20. The ovalbumin-specific T-cell-T-cell hybridomas, DO-11.10 and 3DO-54.8, were used to detect processed antigen. These T-T hybridomas have different fine specificities for the p323-339 determinant. A20 cells presented native ovalbumin well to both T-T hybridomas, whereas M12 cells presented native ovalbumin well to 3DO-54.8 but very inefficiently to DO-11.10. M12 and A20 cells effectively stimulated both T-T hybridomas with the same concentrations of the immunogenic synthetic peptide p323-339. Therefore, M12 cells and DO-11.10 can interact with each other, and both T-T hybridomas have similar sensitivities for the same immunogenic peptide. We conclude that genetically identical antigen-presenting cells can display heterogeneity in the fine processing of an immunodominant T-cell determinant, and synthetic model peptides that represent the minimal stimulatory sequence of a T-cell determinant are not necessarily identical to the structure of in vivo processed antigen. Heterogeneity in antigen processing by individual antigen-presenting cells would serve to increase the repertoire of immunogenic peptides that are presented to T cells.

Animals↗
PubMed1988

Effect of gangliosides on activation of the alternative pathway of human complement.

Liposomes as defined model membranes were used to quantitatively study the effects of specific sialic acid containing glycolipids on activation of the alternative pathway of human C. Liposomes containing dimyristoylphosphatidylethanolamine, cholesterol, and cerebrosides at molar ratios of 1.0/0.75/0.33 activated the alternative pathway in human serum treated with MgEGTA. Activation was measured by C3 conversion and the deposition of total C3 and functional C3b on the liposome surface. The monosialoganglioside GM1, when incorporated into the activating liposome membrane at molar ratios between 10(-5) and 10(-2), inhibited activation in a dose-dependent manner. Sialosylparagloboside also inhibited activation in human serum, and inhibition was completely reversed after neuraminidase treatment. The degree of inhibition by GM1 correlated with the relative amount of GM1 exposed on the liposome surface. Sialic acid did not directly inhibit the binding of C3b when liposomes containing gangliosides were incubated with the purified components C3, B, D, and P. GM1 did inhibit activation when liposomes were incubated with a mixture of purified C3, B, D, P, H, and I. Binding assays with radiolabeled H showed increased binding of H to liposome-bound C3b in the presence of GM1. These results establish the ability of sialic acid on glycolipids to promote H binding to C3b and thereby regulate alternative pathway activation on a defined lipid membrane.

Binding, Competitive↗
PubMed1988

Inhibition of the alternative pathway of human complement by structural analogues of sialic acid.

Liposomes were used to determine whether gangliosides containing certain structurally defined analogues of sialic acid could inhibit activation of the alternative pathway of human C. Gangliosides containing sialic acid residues with modifications in the N-acetyl group, carboxyl group, or polyhydroxylated tail were either isolated from natural sources or prepared by chemical modification of the native sialic acid structure. Sialic acid lost more than 90% of its inhibitory activity after removal of just the C9 carbon from the polyhydroxylated tail. Sialic acid was also unable to inhibit activation after converting the carboxyl group to a hydroxymethyl group. Galactose oxidase/NaB3H4 treatment of liposomes containing gangliosides with native or modified sialic acid residues confirmed that neither modification altered the amount of gangliosides exposed at the liposome surface. Changing the N-linked acetyl group to a glycolyl group had no effect on the inhibitory activity of sialic acid. These data further define the structural features of sialic acid that are important in regulation of alternative pathway activation. Both the C9 carbon of the polyhydroxylated tail and the carboxyl group are essential for this function; whereas, the N-linked acetyl group may be modified without loss of activity.

Binding, Competitive↗
PubMed1981

Feline leukemia - unique and cross-relating antigens on individual virus-producing tumors identified by complement-dependent antibody.

Following exposure to feline leukemia virus (FeLV), cats that resist leukemia produce complement dependent antibodies (CDA) which lyse leukemia cells in the presence of cat complement. The CDA activity has been shown to be directed to feline oncornavirus-associated cell membrane antigen (FOCMA) found on the surface of most feline leukemias. Despite the retrovirus etiology of feline leukemia, and the ubiquity of FOCMA expression by individual tumors, we found that CDA activity did not cross-react when five feline leukemia lines were compared (FL74, F422, 3272, 3281, and 79-14940). Profiles for CDA mediated lysis of the leukemia target lines were determined for sera from 149 FeLV exposed cats, and animals were identified whose sera would selectively lyse almost any possible combination of tumors from one to five lines tested. Cross absorption and cold target inhibition studies were performed on selected sera, and a spectrum of different antigens was identified which included both individual determinants and antigens shared by some of the tumor lines. Leukemias FL74 and F422 carried a cross-reactive determinant which was distinct from a second antigen shared by 3272 and 3281; lines F422, 3272 and 3281 each also carried a unique antigen. Leukemia 79-14940 did not cross-react with other target tumor lines. Targets of CDA mediated lysis on leukemia cells maybe associated with normal feline cell surface antigens, because CDA titers were sometimes reduced after serum absorption with normal cat spleen cells. The relevance of multiple FOCMA determinants is discussed in terms of tumor escape from immunity.

Animals↗
PubMed1980

Protection of cats against progressive fibrosarcomas and persistent leukemia virus infection by vaccination with feline leukemia cells.

Young cats (3-6 mo old) were challenged with oncogenic Snyder-Theilen feline sarcoma virus (FeSV) after vaccination with live or killed FL74 cat lymphoma cells. Compared with controls immunized with normal cat fibroblasts, the FL74-vaccinated cats exhibited increased resistance to FeSV-induced progressive primary and disseminated secondary tumors. Maximum protection was achieved by vaccination with live FL74 cells or with a low dose of freeze-thawed cells, but tumor cells inactivated by glutaraldehyde or paraformaldehyde were also effective. Infectious helper feline leukemia virus (FeLV) was detected in the blood of all cats after FeSV challenge, but the duration and magnitude of this viremia were reduced in animals that had been previously vaccinated with live, freeze-thawed, or paraformaldehyde-fixed cells. Although immunized cats were resistant to FeSV-induced tumors and FeLV viremia, no evidence was obtained to suggest that vaccination with dead cells induced detectable circulating antibody prior to challenge with oncogenic virus. After FeSV challenge, complement-dependent antibody to feline oncornavirus-associated cell membrane antigen (CDA-FOCMA) appeared at high titer in cats that were destined either to survive tumor-free or to develop small, localized, and eventually regressing tumors. Cats immunized with live FL74 cells developed CDA-FOCMA prior to challenge, and antibody appeared in these cats following an episode of transient FeLV viremia induced by virus replicating from the injected tumor cells. Therefore, apparently, a state of transient or persistent FeLV viremia regularly preceded detection of CDA-FOCMA activity. Several individually derived feline lymphoma cell lines were used as targets for CDA-FOCMA, and the results suggested that lytic activity is directed to multiple antigen determinants expressed differently by individual feline lymphomas.

Animals↗