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S K Pierce

Publications and source records attributed to S K Pierce.

At least 37 records · Page 2Linked to original sources
PubMed1999

A role for lipid rafts in B cell antigen receptor signaling and antigen targeting.

The B cell antigen receptor (BCR) serves both to initiate signal transduction cascades and to target antigen for processing and presentation by MHC class II molecules. How these two BCR functions are coordinated is not known. Recently, sphingolipid- and cholesterol-rich plasma membrane lipid microdomains, termed lipid rafts, have been identified and proposed to function as platforms for both receptor signaling and membrane trafficking. Here we show that upon cross-linking, the BCR rapidly translocates into ganglioside G(M1)-enriched lipid rafts that contain the Src family kinase Lyn and exclude the phosphatase CD45R. Both Igalpha and Lyn in the lipid rafts become phosphorylated, and subsequently the BCR and a portion of G(M1) are targeted to the class II peptide loading compartment. Entry into lipid rafts, however, is not sufficient for targeting to the antigen processing compartments, as a mutant surface Ig containing a deletion of the cytoplasmic domain is constitutively present in rafts but when cross-linked does not internalize to the antigen processing compartment. Taken together, these results provide evidence for a role for lipid rafts in the initial steps of BCR signaling and antigen targeting.

Animals↗
PubMed1999

MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens.

Processing and presentation by Ag-specific B cells is initiated by Ag binding to the B cell Ag receptor (BCR). Cross-linking of the BCR by Ag results in a rapid targeting of the BCR and bound Ag to the MHC class II peptide loading compartment (IIPLC). This accelerated delivery of Ag may be essential in vivo during periods of rapid Ag-driven B cell expansion and T cell-dependent selection. Here, we use both immunoelectron microscopy and a nondisruptive protein chemical polymerization method to define the intracellular pathway of the targeting of Ags by the BCR. We show that following cross-linking, the BCR is rapidly transported through transferrin receptor-containing early endosomes to a LAMP-1+, beta-hexosaminadase+, multivesicular compartment that is an active site of peptide-class II complex assembly, containing both class II-invariant chain complexes in the process of invariant chain proteolytic removal as well as mature peptide-class II complexes. The BCR enters the class II-containing compartment as an intact mIg/Igalpha/Igbeta complex bound to Ag. The pathway by which the BCR targets Ag to the IIPLC appears not to be identical to that by which Ags taken up by fluid phase pinocytosis traffick, suggesting that the accelerated BCR pathway may be specialized and potentially independently regulated.

Animals↗
PubMed1999

Regulation of B cell receptor-mediated MHC class II antigen processing by FcgammaRIIB1.

The processing and presentation of Ag by Ag-specific B cells is highly efficient due to the dual function of the B cell Ag receptor (BCR) in both signaling for enhanced processing and endocytosing bound Ag. The BCR for IgG (FcgammaRIIB1) is a potent negative coreceptor of the BCR that blocks Ag-induced B cell proliferation. Here we investigate the influence of the FcgammaRIIB1 on BCR-mediated Ag processing and show that coligating the FcgammaRIIB1 and the BCR negatively regulates both BCR signaling for enhanced Ag processing and BCR-mediated Ag internalization. Treatment of splenic B cells with F(ab')2 anti-Ig significantly enhances APC function compared with the effect of whole anti-Ig; however, whole anti-Ig treatment is effective when binding to the FcgammaRIIB1 was blocked by a FcgammaRII-specific mAb. Processing and presentation of Ag covalently coupled to anti-Ig were significantly decreased compared with Ag coupled to F(ab')2anti-Ig; however, the processing of the two Ag-Ab conjugates was similar in cells that did not express FcgammaRIIB1 and in splenic B cells treated with a FcgammaRII-specific mAb to block Fc binding. Internalization of monovalent Ag by B cells was reduced in the presence of whole anti-Ig as compared with F(ab')2 anti-Ig, but the internalized Ag was correctly targeted to the class II peptide loading compartment. Taken together, these results indicate that the FcgammaRIIB1 is a negative regulator of the BCR-mediated Ag-processing function.

Animals↗
PubMed1999

CD19 regulates B cell antigen receptor-mediated MHC class II antigen processing.

In B cells, the processing of antigens in the context of MHC class II molecules is initiated by the binding of antigen to the B cell antigen receptor (BCR). The BCR serves two roles in antigen processing, signaling for enhanced processing and endocytosing bound antigen. CD19 is a B cell surface molecule which has been demonstrated to function in modifying signals generated through the BCR, regulating T-cell dependent B-cell activation. Here we provide evidence that cross-linking CD19 selectively blocked BCR-mediated enhancement of the processing and presentation of antigens taken up by fluid pinocytosis. CD19 cross-linking also inhibited the processing and presentation of antigen internalized bound to the BCR by decreasing the degree and rate of internalization of the BCR and specific antigen and its trafficking to the class II peptide loading compartment. In contrast, CD19 cross-linking did not affect the rate of assembly of SDS-stable peptide class II complexes, indicating that CD19 cross-linking did not have a global effect on membrane trafficking in B cells but rather a selective effect on BCR trafficking. Thus, in addition to a direct role in modulating BCR signaling for B cell proliferation and differentiation, CD19 may indirectly influence B cell activation by regulating antigen processing and B cell interactions with helper T cells.

Animals↗
PubMed1998

The assembly and stability of MHC class II-(alpha beta)2 superdimers.

X-ray crystallography of several MHC class II molecules revealed a structure described as a dimer of heterodimers, or a superdimer. This discovery led to the hypothesis that MHC class II molecules may interact with the TCR and CD4 as an (alpha beta)2 superdimer, potentially providing more stable and stimulatory interactions than can be provided by the simple alpha beta heterodimer alone. In this study, using chemical cross-linking, we provide evidence for the existence of the superdimers surface of B cells. We further characterize the superdimers and demonstrate that in lysates of B cells, I-Ek dimers and superdimers are derived from the same population of I-Ek molecules. Purified, I-Ek molecules in solution also exist as a mixture of 60-kDa dimers and 120-kDa superdimers, indicating that I-Ek has an intrinsic ability to form 120-kDa complexes in the absence of other cellular components. Peptide mapping showed that the alpha beta and (alpha beta)2 complexes are closely related and that the superdimers do not contain additional polypeptides not present in the dimers. The (alpha beta)2 complex displays thermal and pH stability similar to that of the alpha beta complex, both being denatured by SDS at temperatures above 50 degrees C and at a pH below 5. These data support the model that MHC class II has an intrinsic ability to assume the (alpha beta)2 superdimeric conformation, which may be important for interactions with the TCR and CD4 coreceptor.

Animals↗
PubMed1998

Signaling through the B cell antigen receptor regulates discrete steps in the antigen processing pathway.

Antigen processing in B cells is initiated by antigen binding to the surface B cell antigen receptor (BCR). The BCR is a signaling receptor which also functions to endocytose bound antigen for subsequent intracellular processing and presentation with class II molecules. Previously, using subcellular fractionation, we showed that although the surface BCR constitutively traffics from the cell surface to the class II peptide-loading compartment (IIPLC), cross-linking the BCR regulates trafficking, resulting in a more rapid movement of the BCR to the IIPLC (Song et al., 1995, J. Immunol. 155, 4255). The rate of degradation of both the BCR and the bound antigen was also accelerated following BCR cross-linking. Here we provide evidence that the effect of cross-linking the BCR on antigen processing is in part dependent on signal cascades initiated by the BCR. We show that the protein kinase inhibitors Genistein and Chelerythrine, which block BCR signaling, reduce BCR-enhanced antigen processing in a dose-dependent manner. The kinase inhibitors have a small effect on the rate of internalization of the BCR and antigen following BCR cross-linking and significantly decrease the accelerated trafficking to the IIPLC. The increased rate of degradation of the BCR and antigen induced by BCR cross-linking is also decreased by the kinase inhibitors. BCR signaling does not appear to have a global effect on intracellular membrane trafficking as cross-linking the BCR did not alter the rate of trafficking of newly synthesized class II molecules to the IIPLC. Thus, the signaling function of the BCR appears to play a significant role in regulating discrete steps in the intracellular antigen processing pathway.

Alkaloids↗
PubMed1997

Wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocks the assembly of peptide-MHC class II complexes.

Peptide-class II complexes are assembled in endocytic, lysosome-like compartments where newly synthesized class II molecules are targeted from the trans-Golgi network (TGN). Recent studies have implicated phosphatidylinositol 3-kinase (PI3-kinase) as an essential component in membrane trafficking from the TGN to lysosomes. Here, using subcellular fractionation, we show PI3-kinase activity associated with subcellular fractions which contain the class II peptide-loading compartment (IIPLC) in B cells. At concentrations required for inhibition of PI3-kinase activity in vivo, wortmannin blocked the processing and presentation of antigen by B cells to T cells. Treatment of B cells with wortmannin significantly limited the proteolytic degradation of invariant chain and the formation of peptide-class II complexes. Subcellular fractionation coupled with pulse-chase analyses showed that invariant chain and class II molecules trafficked to the IIPLC in wortmannin-treated cells. However, wortmannin prevented the maturation and correct targeting to the IIPLC of cathepsin D, a protease necessary for the degradation of invariant chain and assembly of processed antigen-class II complexes. These results suggest that li-class II complexes traffic to the IIPLC via a pathway that is relatively insensitive to wortmannin, but suggest a role for PI3-kinases in the trafficking of other components necessary for the assembly of processed antigen class II complexes to the IIPLC.

Androstadienes↗
PubMed1996

HLA-DM is present in one-fifth the amount of HLA-DR in the class II peptide-loading compartment where it associates with leupeptin-induced peptide (LIP)-HLA-DR complexes.

HLA-DM has been shown in vitro to catalyze the release of invariant chain (Ii) derived peptides from the peptide-binding groove of class II molecules, thereby facilitating the binding of antigenic peptides. Previous studies showed that at steady state, the majority of DM resides in the class II peptide-loading compartment (IIPLC) where Ii dissociates from class II molecules and antigenic peptides are bound. Here we characterize the expression of DM in vivo in subcellular fractions containing the IIPLC. Using quantitative immunoblotting, we show that in the cell as a whole, class II molecules are expressed in 23-fold molar excess of DM. However, DM is concentrated in the IIPLC, where it is present in a considerably higher concentration relative to the class II molecules, in a molar ratio of 5DR:1 DM. This molar ratio of DM to DR in the IIPLC in vivo is consistent with the catalytic function proposed for DM from studies in vitro. We also provide both biochemical and genetic evidence that DM associates with complexes which contain Ii fragments and class II molecules in the IIPLC. Such complexes are only observed in leupeptin-treated cells in which Ii fails to be completely degraded and complexes containing the leupeptin-induced fragment of Ii (LIP) and class II molecules accumulate in the IIPLC. This observation is consistent with LIP-class II complexes being a substrate for DM in vivo and suggests that interactions of DM and LIP-class II are extremely transient under normal conditions.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1996

Chloroplast genes are expressed during intracellular symbiotic association of Vaucheria litorea plastids with the sea slug Elysia chlorotica.

The marine slug Elysia chlorotica (Gould) forms an intracellular symbiosis with photosynthetically active chloroplasts from the chromophytic alga Vaucheria litorea (C. Agardh). This symbiotic association was characterized over a period of 8 months during which E. chlorotica was deprived of V. litorea but provided with light and CO2. The fine structure of the symbiotic chloroplasts remained intact in E. chlorotica even after 8 months of starvation as revealed by electron microscopy. Southern blot analysis of total DNA from E. chlorotica indicated that algal genes, i.e., rbcL, rbcS, psaB, psbA, and 16S rRNA are present in the animal. These genes are typically localized to the plastid genome in higher plants and algae except rbcS, which is nuclear-encoded in higher plants and green (chlorophyll a/b) algae. Our analysis suggests, however, that similar to the few other chromophytes (chlorophyll a/c) examined, rbcS is chloroplast encoded in V. litorea. Levels of psbA transcripts remained constant in E. chlorotica starved for 2 and 3 months and then gradually declined over the next 5 months corresponding with senescence of the animal in culture and in nature. The RNA synthesis inhibitor 6-methylpurine reduced the accumulation of psbA transcripts confirming active transcription. In contrast to psbA, levels of 16S rRNA transcripts remained constant throughout the starvation period. The levels of the photosystem II proteins, D1 and CP43, were high at 2 and 4 months of starvation and remained constant at a lower steady-state level after 6 months. In contrast, D2 protein levels, although high at 2 and 4 months, were very low at all other periods of starvation. At 8 months, de novo synthesis of several thylakoid membrane-enriched proteins, including D1, still occurred. To our knowledge, these results represent the first molecular evidence for active transcription and translation of algal chloroplast genes in an animal host and are discussed in relation to the endosymbiotic theory of eukaryote origins.

Animals↗
PubMed1996

B cell antigen receptor signaling links biochemical changes in the class II peptide-loading compartment to enhanced processing.

In B cells, processing of antigens in the context of MHC class II molecules is initiated by the binding of antigen to the B cell antigen receptor (BCR). BCR-mediated processing is highly efficient, as a consequence of the BCR's linked roles of delivering antigen to the class II peptide-loading compartment and of signaling for increased antigen-processing activity. Evidence is emerging that receptor signaling regulates intracellular transport through the activities of kinases. These in turn have been implicated in the regulation of small mol. wt GTPases which govern membrane transport. Therefore, we investigated the changes in the phosphoprotein and GTPase profiles associated with the class II peptide-loading compartment following BCR cross-linking. We first show that protein kinase inhibitors, known to block BCR signal transduction, inhibit BCR-enhanced antigen processing, demonstrating the critical dependence of enhanced processing on the signaling activity of the BCR. Consistent with this observation, the phosphoprotein profile of the class II peptide-loading compartment underwent rapid and transient changes following BCR cross-linking. We also observed a marked increase in the low mol. wt GTPases associated with the class II peptide-loading compartment within 5 min of BCR cross-linking. The observed changes in both the phosphoprotein and GTPase profiles associated with the peptide-loading compartment were blocked by kinase inhibitors and were not accompanied by overall gross changes in the protein composition of the subcellular compartments. Thus, signal cascades initiated by BCR cross-linking at the plasma membrane are translated into changes in specific subsets of regulatory proteins associated with the peptide-loading compartment.

Animals↗
PubMed1996

Class II antigen processing compartments and the function of HLA-DM.

The DM alpha and DM beta genes encode a nonpolymorphic, class II-like molecule which functions by an, as yet, undefined mechanism in the assembly of Major Histocompatibility Complex class II-peptide complexes. Indeed, mutant cells which express class II molecules but fail to express DM are unable to process and present native protein antigens. A striking phenotype of the mutation is class II molecules that contain almost exclusively a nested set of invariant chain peptides, termed CLIP, for class II associated Ii peptides, instead of the normal array of endogenously and exogenously derived peptides. Thus, DM appears to be required for the correct assembly of processed antigen-class II complexes. Recently, the subcellular compartments that contain DM and in which functional processed antigen-class II complexes are first formed have been described. Here, the evidence for the function of DM in the antigen-processing compartments is reviewed.

Antigen Presentation↗
PubMed1995

Entry of B cell antigen receptor and antigen into class II peptide-loading compartment is independent of receptor cross-linking.

The processing and presentation of Ag by B lymphocytes are initiated by Ag binding to the B cell Ag receptor (BCR). Using subcellular fractionation, we recently identified a compartment in B cells in which functional, processed Ag-class II complexes are formed following BCR-mediated Ag internalization, referred to as the peptide-loading compartment. These studies, however, did not address the transport of Ag or BCR from the cell surface to the peptide-loading compartment. In this work, we describe the intracellular trafficking of Ag and surface Ig (sIg) in B cells and evaluate the effect of cross-linking sIg on this intracellular movement. We show that sIg constitutively transports Ag from the plasma membrane, through endosomes, to the MHC class II peptide-loading compartment. The cross-linking of the BCR increases the rate of internalization of sIg and bound Ag, but does not alter the trafficking pathway. Thus, the delivery of Ag to the class II peptide-loading compartment by the sIg is independent of BCR cross-linking, but can be influenced by BCR cross-linking.

Animals↗
PubMed1995

The intracellular transport of MHC class II molecules in the absence of HLA-DM.

The HLA-DM alpha and HLA-DM beta genes encode a nonpolymorphic, class II-like molecule that functions by an as yet undefined mechanism in the assembly of processed antigen-HLA class II complexes. Mutant cells that fail to express HLA-DM are deficient in Ag processing. We previously isolated a subcellular compartment in mouse B cells in which functional processed Ag-class II complexes are first formed, referred to as the peptide-loading compartment. Here, evidence is provided that HLA-DM resides in a subcellular compartment with the characteristics of a peptide-loading compartment in a human B lymphoblastoid cell line, but is not required for the intracellular transport of HLA-DR3 molecules to a corresponding compartment in HLA-DM-deficient cells. Thus, the primary defect in HLA-DM-deficient cells does not appear to be a failure in the intracellular trafficking of class II molecules.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1995

Intracellular transport of invariant chain-MHC class II complexes to the peptide-loading compartment.

Th cells recognize peptide fragments of foreign Ags bound to MHC class II molecules. Upon synthesis in the endoplasmic reticulum, the alpha- and beta-chains of the class II molecules rapidly associate with invariant chains (li). The dissociation of li from class II molecules precedes binding of processed Ag and the formation of SDS-stable alpha beta dimers. We previously showed that functional, processed Ag-class II complexes are assembled in a dense lysosome-like compartment that contains stable class II molecules, but no li, referred to in this work as the peptide-loading compartment. We also identified a separate compartment that contains predominantly SDS-unstable li-class II complexes. Because we were unable to identify known organelle markers associated with this compartment, we refer to it as the X compartment. In this work, we provide results that indicate that the X compartment is composed of transport vesicles that move li-class II complexes to the peptide-loading compartment, where all events in the assembly of processed Ag-class II complexes occur.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1995

Cross-linking cell surface class II molecules stimulates Ig-mediated B cell antigen processing.

Th cells bind to peptide-class II complexes presented on B cell surfaces. Recent evidence indicates that upon cross-linking, class II molecules transduce signals that modulate a variety of B cell functions. One possible function of class II signaling is to regulate the assembly of processed Ag-class II complexes. Here we show that cross-linking B cell surface class II molecules augments the processing and presentation of an Ek-restricted Ag to a specific T cell hybrid. Significantly, class II cross-linking only affects processing initiated by Ag binding to the surface Ig. The processing of Ag taken up by fluid phase pinocytosis is not affected by class II cross-linking, nor is the presentation of an antigenic peptide that does not require processing. Augmentation of Ag processing is enhanced by treatment of B cells with dibutyryl cAMP, a second messenger in the class II signaling pathway. The cross-linking of class II molecules does not alter the rate or number of Ig molecules internalized or the biosynthesis or expression of Ek molecules. Moreover, changes in the expression of the B7 family of costimulatory molecules or the adhesion molecule LFA-1 (CD11a/CD18) induced by class II cross-linking do not appear to account for the augmentation of processing observed here. Thus, the cross-linking of class II molecules on B cell surfaces selectively stimulates Ig-mediated Ag processing, indicating that a step in this pathway is a target of class II-mediated signaling events.

Animals↗
PubMed1995

CD40-CD40 ligand interactions stimulate B cell antigen processing.

The interactions between B cell CD40 and T cell CD40 ligand (CD40L) have been shown recently to play an important role in T cell-dependent activation of B cells. Here, we show that the ligation of CD40 stimulates the processing of antigen by B cells. The activation of an antigen-specific T cell hybrid by B cells co-cultured with insect cells expressing recombinant CD40L or with a CD40-specific monoclonal antibody requires less antigen and fewer B cells compared to control cells. The augmentation was observed both for processing initiated by antigen binding to and cross-linking the surface immunoglobulin, and processing of antigen taken up by fluid-phase pinocytosis. CD40 appears to affect a step in the intracellular processing of antigen, as CD40 has no effect on the presentation of an antigenic peptide which does not require processing. In addition, the CD40-induced augmentation of processing is not attributable to the effect of CD40 ligation on the cell surface expression of B7, LFA-1 or CD23. CD40 ligation does not affect the biosynthesis of the class II EK molecules, and although ligation of CD40 induces B cell proliferation, the augmentation of processing does not require proliferation. The ability of CD40 to stimulate B cell antigen processing has the potential to influence significantly the outcome of antigen-dependent T cell-B cell interactions.

Adjuvants, Immunologic↗
PubMed1995

The structure of MHC class II: a role for dimer of dimers.

The MHC class II molecules, expressed by antigen presenting cells, are heterodimers composed of an alpha and a beta chain, which function to present processed antigen to helper T cells. The human MHC class II molecules, HLA-DR1 and HLA-DR3, crystallized not as monomers, but rather dimers of alpha beta heterodimers. The 'dimer of dimers' or 'superdimer' structure led to speculation that the binding of T-cell receptors to monomeric class II molecules on the antigen presenting cell surface may affect dimerization and thus initiate signaling both in the T cell and in the antigen presenting cell. Recent biochemical analyses of the mouse MHC class II Ek molecule provide evidence that dimers of class II heterodimers form in the absence of T cells. Although such dimers were shown to augment T-cell stimulation, the dimerization of class II molecules alone is unlikely to initiate signal transduction. However, dimers may be important in stabilizing weak T-cell receptor/CD4/class II interactions, allowing further multimerization of such complexes, leading to signaling.

Animals↗