Peptide Alpha Research Articles

Identification of a region in the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX complex that binds to purified actin-binding protein.

The platelet membrane glycoprotein (GP) Ib-IX complex is a major site of attachment of the platelet membrane skeleton to the plasma membrane. This association is mediated by the interaction of actin-binding protein with the GP Ib-IX complex. The aim of the present work was to identify domains on the GP Ib-IX complex that interact with actin-binding protein. Synthetic peptides corresponding to sequences of the GP Ib alpha-chain and beta-chain cytoplasmic domains were analyzed for their ability to bind to purified actin-binding protein. Two overlapping peptides encompassing a sequence (Thr-536-Phe-568) from the central region of the cytoplasmic domain of GP Ib alpha were the most effective in binding 125I-actin-binding protein, as assessed by a microtiter well approach and peptide affinity chromatography. One of the active peptides (Thr-536-Leu-554) was chosen to evaluate the likelihood that the central region of the cytoplasmic domain of GP Ib alpha is involved in binding of the intact complex to actin-binding protein. This peptide could be specifically cross-linked to purified actin-binding protein in solution. Rabbit polyclonal antibody against this peptide inhibited the binding of purified actin-binding protein to the purified GP Ib-IX complex. Finally, as in intact platelets, the calpain-induced hydrolytic fragments of purified actin-binding protein (M(r) = 200,000 and M(r) = 91,000) showed little binding to the GP Ib alpha peptide. Taken together, these results provided evidence that a region between Thr-536 and Phe-568 of the cytoplasmic domain of GP Ib alpha participates in the interaction of the GP Ib-IX complex with actin-binding protein.

Two subsets of epithelial cells in the thymic medulla.

Information was sought on the features of epithelial cells in the murine thymic medulla. The expression of major histocompatibility complex (MHC) molecules on medullary epithelium was defined by light microscopy with the aid of bone marrow chimeras and MHC-transgenic mice. A proportion of medullary epithelial cells was found to show conspicuously high expression of conventional MHC (H-2) class I (K, D, L) and class II (I-A, I-E) molecules. These cells express a high density of the Y-Ae epitope, a complex of an E alpha peptide and I-Ab molecules found on typical bone marrow-derived cells. MHC+ medullary epithelial cells show limited expression of I-O molecules, a class of atypical nonpolymorphic MHC-encoded class II molecules present on B cells. Other medullary epithelial cells express a high density of I-O molecules but show little or no expression of typical MHC class I or II molecules. MHC and I-O expression thus appear to subdivide medullary epithelial cells into two phenotypically distinct subsets. This applies in adults. In the embryonic thymus most medullary epithelial cells express both types of molecules.

Interleukin 1: the patterns of translation and intracellular distribution support alternative secretory mechanisms.

Interleukin-1 (IL-1) is synthesized as a 31 kDa precursor protein, whose multiple extracellular activities are attributed to receptor binding of a processed, carboxy-terminal 17 kDa peptide. Unlike other secreted proteins, the IL-1 precursor lacks a hydrophobic leader sequence and is not found in organelles composing the classical secretory pathway. In order to further clarify the intracellular processing of IL-1, we studied its site of synthesis in human monocytes. Secreted and integral membrane proteins are translated on membrane-bound polyribosomes, while intracellular proteins are translated on free polyribosomes. Free and membrane-bound polysomes were isolated from Lipid A-stimulated monocyte lysates and immunoblotted using antibodies specific to the N-terminal regions of the IL-1 alpha and beta precursors. Free polysome fractions showed multiple small bands consistent with nascent peptide chains; membrane-bound polysomes yielded no detectable IL-1. Polysome fractions were then analyzed by immunoelectron microscopy; nascent IL-1 alpha and beta peptide chains were readily seen emerging from cytoskeletal-associated free polyribosomes, but not membrane-bound polyribosomes. Electron microscopic in situ hybridization revealed IL-1 mRNA chains attached to cytoskeletal-associated free, but not membrane-bound polyribosomes. The intracellular distribution of the fully synthesized IL-1 beta precursor was studied in human mesangial cells (HMC), whose cytoskeletal organization is more readily evaluated than that of monocytes. Dual immunofluorescence microscopy of these cells revealed a complex intracellular distribution of the fully synthesized 31 kDa IL-1 precursors. IL-1 was asymmetrically distributed between cytosolic, microtubule, and nuclear compartments, without association with actin or intermediate filaments. This demonstration of the sites of IL-1 synthesis and patterns of intracellular distribution provide further evidence for an extracellular release mechanism which is clearly distinct from the classical secretory pathway.

Epidermal growth factor dependence and TGF alpha autocrine growth regulation in primary rat tracheal epithelial cells.

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGF alpha autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGF alpha peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGF alpha mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGF alpha antiserum and by a tyrphostin compound that blocks TGF alpha/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGF alpha as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation.

Beta-hexosaminidase splice site mutation has a high frequency among non-Jewish Tay-Sachs disease carriers from the British Isles.

In the course of defining mutations causing Tay-Sachs disease (TSD) in non-Jewish patients and carriers from the British Isles, we identified a guanine to adenine change (also previously described) in the obligatory GT sequence of the donor splice site at the 5' end of intron 9 of the hexosaminidase alpha peptide gene. Of 24 unrelated mutant chromosomes from 20 non-Jewish subjects (15 TSD carriers, four TSD patients, and one TSD fetus), five had mutations common in the Ashkenazi Jewish community, and 10 had the intron 9 splice site mutation. This is an unexpected result considering the diverse origin of the population of the British Isles. This mutation was not found in 28 control UK subjects or 11 Jewish carriers of known TSD mutations. Before attempting detection of unknown mutations, non-Jewish TSD carriers from the British Isles should be screened for the intron 9 donor splice site mutation as well as those mutations which predominate in the Jewish community.

Localization of the cross-linking sites of RGD and KQAGDV peptides to the isolated fibrinogen receptor, the human platelet integrin glycoprotein IIb/IIIa. Influence of peptide length.

The non-covalent and Ca(2+)-dependent heterodimer GPIIb/IIIa, formed by platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa), also known as the integrin alpha IIb beta 3, is the inducible receptor for fibrinogen and other adhesive proteins on the surface of activated platelets. A fraction of the isolated GPIIb/IIIa in solution binds RGD or KQAGDV inhibitory peptides and, upon peptide removal, apparently acquires the capacity to bind fibrinogen ('activated' GPIIb/IIIa) [Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C. & Ginsberg, M. H. (1991) Cell 65, 409-416]. Photoaffinity labelling was used here to study the ligand binding site(s) of GPIIb/IIIa in solution, for which the peptides CKRKRKRKRRGDV (alpha 1), CGRGDF (alpha 2), CYHHLGGAKQAGDV (gamma 1) and CGAKQAGDV (gamma 2) were synthesized with a photoactivable cross-linker group and a fluorescent reporter group attached to the N-terminal cysteine residue. Contrary to the situation in activated platelets, both GPIIb and GPIIIa were equally labelled by the four peptides and the cross-linking sites were localized by protein chemical analyses of the fluorescently labelled tryptic peptides of both subunits. Thus, the localization of the cross-linking sites in GPIIb varies considerably with the peptide length and is very different from that localization observed in activated platelets: alpha 2 and gamma 2 were found cross-linked to the N-terminal of both the heavy (GPIIbH 42-73) and the light (GPIIbL2 30-75) chains of GPIIb; while the longer peptides alpha 1 and gamma 1 were cross-linked to the C-terminal of GPIIbH within the 696-724 and 752-768 peptide stretches, respectively. On the other hand, the cross-linking sites of the four inhibitory peptides in GPIIIa were found mainly within the proteolysis susceptible region, between the N-terminal (GPIIIa 1-52) and the core (GPIIb 423-622) highly disulphide-bonded domains, observing that the longer the peptide the closer the cross-linking site is to the N-terminal of GPIIIa: alpha 1 at GPIIIa 63-87 and 303-350; gamma 1 at GPIIIa 9-37; alpha 2 at GPIIIa 151-191; and gamma 2 at GPIIIa 303-350. These results led us to the following conclusions. (a) The GPIIIa 100-400 region contributes to the ligand-binding domain in GPIIb/IIIa both in solution and in activated platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibody detection of a major self peptide. MHC class II complex.

MHC class I and class II molecules transport foreign and self peptides to the cell surface and present them to T lymphocytes. Detection of these peptide:MHC complexes has thus far been limited to analysis of the response of a T cell. Previously, we showed that a mAb, Y-Ae, reacts with 10 to 15% of class II molecules on peripheral B lymphocytes and on cells in the thymus medulla but not thymus cortex in mice that express both I-Ab and I-Eb molecules. Elsewhere, we show that Y-Ae detects a self E alpha peptide bound to I-Ab molecules. Data presented here suggest that the antibody binds over the peptide binding groove of class II molecules, and, like a TCR, appears to recognize both the self peptide and polymorphic class II residues. In addition to B lymphocytes, the Y-Ae determinant is expressed at comparable levels on other APC, including macrophages and dendritic cells. Finally, the antibody does not react with invariant chain-associated class II complexes, thus providing direct evidence that invariant chain:class II complexes and peptide:class II complexes are mutually exclusive. These data provide further evidence that immunologic self is of limited complexity, and have important implications for T cell selection, self tolerance, and autoreactivity.

Macrophage inflammatory protein 1 modulates macrophage function.

Macrophage inflammatory protein 1 (MIP 1), initially purified from the conditioned medium of endotoxin-stimulated macrophages, is a low m.w. heparin-binding protein doublet comprising two peptides, MIP 1 alpha and MIP 1 beta. Although native doublet MIP 1 has previously been shown to exert pyrogenic, mitogenic, and proinflammatory effects on other cell types, its actions on its cell of origin, the macrophage, have not been well catalogued. Our study reports several aspects of macrophage function that are modulated by MIP 1. MIP 1 was not directly cytotoxic for WEHI tumor cells, but MIP 1-treated macrophage exhibited enhanced antibody-independent macrophage cytotoxicity for tumor targets. MIP 1 treatment stimulated proliferation of mature tissue macrophages, and this effect was enhanced upon costimulations with either CSF-1 or granulocyte-macrophage-CSF. Thioglycollate-elicited peritoneal exudate macrophages incubated with native doublet MIP 1-secreted bioactive TNF and IL-6, as well as immunoreactive IL-1 alpha, and these effects were enhanced significantly when the cells were costimulated with IFN-gamma. Purified preparations of the recombinantly derived MIP 1 alpha peptide alone stimulated the secretion of TNF, IL-1 alpha, and IL-6 by peritoneal macrophages, but MIP 1 beta did not. In fact, as little as eightfold excess MIP 1 beta blocked TNF-induction by MIP 1 alpha to a significant degree. By contrast to these apparent "macrophage activating" properties of MIP 1, the cytokine failed to trigger the macrophage oxidative burst, or to up-regulate the expression of Ia on the macrophage surface. Taken together, these data reveal that MIP 1 peptides act as autocrine modulators of their cells of origin, and raise the possibility that MIP 1 peptides may play a role in modulating macrophage responses to inflammatory stimuli in vivo.

Immunoreactivity of transforming growth factor alpha in the normal adult gastrointestinal tract.

The immunolocalisation of transforming growth factor alpha (TGF alpha) in the normal human oesophagus and the gastrointestinal tract was elucidated using two different antibodies - Ab2, a monoclonal antibody reacting with part of the human TGF alpha molecule, and 26T a rabbit affinity purified polyclonal antibody raised against part of the rat TGF alpha peptide. There have been conflicting reports on the distribution of this growth factor in the gut. The results clearly showed that this peptide regulatory factor is confined to the luminal surface and foveolae in the stomach, restricted to the villous epithelium in the small intestine, and in the colon was seen in the upper one third of the crypt. This pattern indicates that the distribution of this peptide regulatory factor is within the differentiated compartment and indicates a role in differentiation besides its well known proliferative effects.

Myasthenogenicity of a human acetylcholine receptor alpha-subunit peptide: morphology and immunology.

Each of 10 rats inoculated with a synthetic peptide comprising residues 125-147 (without a disulfide bond) of human acetylcholine receptor (AChR) alpha-subunit (H alpha) had deposits of IgG and C3 (immune complexes) and showed morphological changes in the fine structure at the motor end-plates 5 weeks after a single immunization. Antibody to the H alpha peptides was elevated 1 week after immunization, but, antibody levels to solubilized human or rat AChR were very low in 8 of the 10 rats. These results suggest that the immune response to peptide H alpha is the myasthenogenic site, which induces morphological change at the end-plates.

The molecular defect in a family with mild atypical osteogenesis imperfecta and extreme joint hypermobility: exon skipping caused by an 11-bp deletion from an intron in one COL1A2 allele.

We have investigated a family with an autosomal dominantly inherited connective-tissue defect causing extreme joint hypermobility, premature osteoporosis and late-onset fractures. Analysis of collagenous proteins from affected individuals showed a deletion in some alpha 2(I) chains. Peptide mapping localized this to the CB peptide alpha 2CB4, which covers the N-terminal one-third of the protein chain. Polymerase chain reaction amplification and sequencing of cDNA derived from this region of the mRNA identified a heterozygous deletion of the 54 bp comprising exon 9. Similar analysis of the genomic DNA revealed an 11-bp deletion from bp3 to bp13 of IVS-9. This disrupts the consensus 5' splice signal (GTAAGT) and leads to exon skipping. In a family study of 13 affected and unaffected family members using both heteroduplex formation and direct analysis for the deletion, all of the affected, but no unaffected individuals, were found to carry the deletion. This generated a positive Lod score of 2.6 with the Liped programme.

Effects of combination of tumor necrosis factor alpha and chemotactic peptide, f-Met-Leu-Phe, on phagocytosis of opsonized microspheres by human neutrophils.

Pretreatment of normal, human neutrophils with 8 units/ml of TNF-alpha followed by treatment with 10(-8) M FMLP resulted in a synergistic effect of the combination of the two mediators on the enhancement of the phagocytic capacity of the cells. This enhancement of phagocytosis occurred without an additional increase in the upregulation of C3b receptors (CR1) beyond that caused by each mediator alone. Pretreatment of the cells with 8 units/ml of TNF-alpha followed by 10(-6) M FMLP resulted in an additive effect of the mediators on neutrophil phagocytosis, again without an additional up-regulation of CR1. This additive effect resulted in an increase in phagocytic capacity of the neutrophils greater than that obtained by treatment of the cells with 10(-6) M FMLP alone, which heretofore has resulted in the greatest enhancement of phagocytic capacity obtained by any pretreatment condition. These synergistic and additive effects of the combination of mediators could be of great importance in host defense against bacterial infections and have important implications regarding the mechanisms of receptor upregulation and phagocytosis.

T helper function of CD4 + cells specific for defined epitopes on the acetylcholine receptor in congenic mouse strains

We previously identified sequence segments of Torpedo acetylcholine receptor (TAChR) alpha subunit recognized by CD4+ cells of congenic mouse strains of different H-2 haplotypes, susceptible to experimental autoimmune myasthenia gravis. CD4+ cells from BALB/c and CB17 mice (H-2d) recognized the peptide sequences alpha 1-20 and alpha 304-322, while C57BL/6 and BALB/b mice (H-2b) recognized alpha 150-169 and alpha 360-378. C57BL/6 mice recognized to a lesser extent also peptide alpha 181-200. In the present study we demonstrate that CD4+ cells which recognize these epitopes have T-helper function. CD4+ cells from TAChR immunized mice, stimulated in vitro with synthetic epitope peptides, induced proliferation in vitro of B cells via soluble factors which were not strain specific, and induced secretion in vitro of anti-AChR antibodies. Upon in vitro stimulation with T-epitope peptides, they secreted interleukin-2. Immunization of mice with synthetic T-epitope peptides caused sensitization of CD4+ cells, which responded in vitro both to the immunizing peptides and to TAChR, and appearance of anti-AChR antibodies in vivo, further identifying the epitope-specific CD4+ cells as AChR-specific T-helper cells.

Purification and characterization by fast-atom-bombardment mass spectrometry of the polymorphonuclear-leucocyte-elastase-generated Aα (1–21) fragment of fibrinogen from human blood after incubation with calcium ionophore A23187

The stimulation of human blood with a Ca2+ ionophore, A23187, leads to activation of polymorphonuclear leucocytes (PMN) with release of small amounts of catalyticaly active elastase, as demonstrated by the formation of a characteristic product, the N-terminal A alpha (1-21) peptide of the Aa subunit of fibrinogen. The identity of the peptide was initially established by radioimmunoassay (r.i.a.) with an antibody raised to A alpha (1-21). We now provide independent confirmation of the formation of A alpha (1-21) by fast-atom-bombardment-m.s. analysis of the fractions separated chromatographically after spiking of plasma samples with peptide labelled with [2H8]Phe at position 8. Identity of the peptides was established on the basis of their chromatographic retention time and by the distinct peaks in the mass spectra of these fractions. The relative intensities of the molecular ions of natural and labelled peptides were measured. On the basis of a comparison of the peaks of similar intensities, the concentration of the natural peptide at the time of spiking was close (79%) to the amount obtained by r.i.a. An additional peptide, des-alanyl-A alpha (2-21), was also seen. The total amount of material measured by r.i.a. could be accounted for by the sum of these two provides. The addition of label and assay by m.s. has provided an independent physical-chemical method for identifying A alpha (1-21) as a characteristic product of PMN elastase release in whole blood, but which is absent in freshly drawn blood.

Substitution of cysteine for glycine at residue 415 of one allele of the alpha 1(I) chain of type I procollagen in type III/IV osteogenesis imperfecta.

We have examined the type I collagen in a patient with type III/IV osteogenesis imperfecta. Two forms of alpha 1(I) chain were produced, one normal and the other containing a cysteine residue within the triple helical domain of the molecule. Cysteine is not normally present in this domain of type I collagen. Peptide mapping experiments localised the mutation to peptide alpha 1(I)CB3 which spans residues 403 to 551 of the triple helix. Subsequent PCR amplification of cDNA covering this region followed by sequencing showed a G to T single base change in the GGC codon for glycine 415 generating TGC, the codon for cysteine. The effect of the mutation on the protein is to delay secretion from the cell, reduce the thermal stability of the molecule by 2 degrees C, and cause excessive post-translational modification of all chains in molecules containing one or more mutant alpha 1(I) chains. The clinical phenotype observed in this patient and the position of the mutation conform to the recent prediction of Starman et al that Gly----Cys mutations in the alpha 1(I) chain have a gradient of severity decreasing from the C-terminus to the N-terminus.

Identification of the retinal cyclic GMP phosphodiesterase inhibitory gamma-subunit interaction sites on the catalytic alpha-subunit.

Retinal rod outer segment phosphodiesterase (PDE) consists of two similar catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). A trypsin-activated soluble PDE exhibiting the ability to be reinhibited by PDE gamma was shown by peptide antisera to retain both N and C termini. Synthetic peptides corresponding to residues 16-30, 78-90, 389-403, and 535-563 of PDE alpha used in a PDE activity assay with trypsin-activated PDE partially prevented inhibition by exogenous PDE gamma; however, only competitions by peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90) were concentration-dependent below 100 nmol of peptide. Binding studies using radio-immunoassays and PDE alpha peptides confirmed that peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90, respectively) were able to bind PDE gamma. Additionally, peptides corresponding to the PDE alpha region 453-534 bound PDE gamma in the binding assay. This suggests that several regions on PDE alpha interact with the PDE gamma inhibitor. While some regions may be involved in binding to PDE gamma, other sites may be involved in PDE gamma inhibition of catalytic activity. Our results suggest that the major regions of PDE alpha that interact with PDE gamma reside within the N terminus (16-30 and 78-90), with weaker interaction regions within or near the hypothesized catalytic domain (453-563). Sequence analysis of three retinal phosphodiesterases (rod outer segment alpha, beta, and cone outer segment alpha') revealed the highest region of dissimilarity in the N and C termini.

Calcitonin gene‐related peptide in the amygdaloid complex of the rat: Immunohistochemical and quantitative distribution, and drug effects on calcium dependent, potassium‐evoked in vitro release

The amygdaloid complex is an area with a high concentration of calcitonin gene-related peptide. In the present paper, immunohistochemical studies revealed a dense innervation of the central nucleus originating most probably from the bed nuclei of the stria terminalis. For determination of tissue concentrations of calcitonin gene-related peptidelike immunoreactivity, the amygdaloid complex was dissected into four parts. The distribution was found to be uneven with the highest concentration (1153.3 fmol/mg protein) in the portion including the nucleus amygdaloideus centralis. High performance liquid chromatographic analysis from an extract of amygdaloid tissue showed that 97% of the calcitonin gene-related peptidelike immunoreactivity measured by radioimmunoassay is authentic rat calcitonin gene-related peptide alpha or beta. Release of calcitonin gene-related peptidelike immunoreactivity was measured in superfused slices of amygdalae pooled from three rats. High potassium (60 mM) caused a significant release of calcitonin gene-related peptidelike immunoreactivity (from 0.88% of total tissue content to 1.91%) from the amygdaloid complex in vitro, which was blocked in calcium-free buffer. Pretreatment with haloperidol or clozapine caused a significant reduction of the 60 mM potassium-evoked release, compared with a saline treated control group (control 21.0 fmol; haloperidol 2.8 fmol; clozapine 8.8 fmol) and an increase of tissue levels after haloperidol treatment by 43%. These results demonstrate that calcitonin gene-related peptide is integrated in amygdaloid functions and possibly a target for actions of neuroleptic drugs.

Characterization of a cDNA clone encoding molluscan insulin‐related peptide II of Lymnaea stagnalis

A cDNA clone encoding molluscan insulin-related peptide (MIP) II was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP cDNA (renamed MIP-I cDNA). The MIP-II cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and A and B chains; however, in this case connected by two distinct C peptides, C alpha and C beta, instead of a single C peptide, a phenomenon which represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIP II and I differ remarkably in primary structure; in contrast, the C alpha peptide domains are fully identical. MIP II has only limited sequence similarity with insulins and related peptides. Both MIP II and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I and II genes are expressed in a single type of neuron: the growth-controlling neuroendocrine light green cells of the Lymnaea CNS.

T cells that recognize peptide sequences of self MHC class II molecules exist in syngeneic mice.

T cell reactivity toward self MHC class II molecules has been recognized in syngeneic MLR in a number of studies, where the T cells are believed to recognize the combination of self/nonself peptide and self MHC molecule. We investigated the stimulation of T cell proliferation by synthetic peptides of sequences corresponding to the first polymorphic amino terminal domain of alpha- and beta-chains of self I-A molecules. Both unprimed and primed T cells responded to a number of peptides of alpha 1 and beta 1 domains of self I-Ad molecules. The response was dependent on the presentation of I-Ad peptides by syngeneic APC and was blocked by anti-class II MHC mAb. Upon further investigation it was observed that I-Ad peptides could inhibit the stimulation of Ag-specific MHC class II-restricted T cell hybridoma due to self presentation of peptides rather than to direct binding of free peptides to the TCR, further supporting their affinity/interaction with intact self MHC class II molecules. The peptide I-A beta d 62-78 showed high affinity toward intact self MHC II molecule as determined by the inhibition of Ag-specific T cell stimulation and yet was nonstimulatory for syngeneic T cells, therefore representing an MHC determinant that may have induced self tolerance. Thus we have shown that strong T cell proliferative responses can be generated in normal mice against the peptides derived from self MHC class II molecules and these cells are part of the normal T cell repertoire. Therefore complete tolerance toward potentially powerful immunodominant but cryptic determinants of self Ag may not be necessary to prevent autoimmune diseases.

Detection of the Na(+)-K(+)-ATPase alpha 3-isoform in multinucleated macrophages

We previously reported that multinucleated macrophages express a high concentration of Na(+)-K(+)-ATPases that are concentrated on the nonadherent domain of their plasma membrane (A. Vignery, T. Niven-Fairchild, D. H. Ingbar, and M. Caplan. J. Histochem. Cytochem. 37: 1265-1271, 1989). We also showed that an increase in newly synthesized alpha-subunit occurred during cell culture and multinucleation. We now present evidence that macrophage multinucleation in vitro is accompanied by an increased accumulation of Na(+)-K(+)-ATPase alpha-subunit mRNA. Most interesting is the detection of significant amount of both alpha 1- and alpha 3-isoform mRNA and peptide in these cells by in situ hybridization, Northern and Western blot analyses. These qualitative and quantitative variations in Na(+)-K(+)-ATPase expression suggest that macrophage multinucleation is accompanied by a coordinated regulation of gene expression and that multinucleation confers a specific function to macrophages. Multinucleated macrophages offer a novel model system to investigate not only the specific function(s) of the alpha 3-isoform but also the role of the Na(+)-K(+)-ATPase in giant cells and osteoclasts.