Abnormal expression of major histocompatibility complex (MHC) class I and class II in various tissues is associated with autoimmune disease. Autoimmune responses can be triggered by viral infections or tissue injuries. We show that the ability of a virus or a tissue injury to increase MHC gene expression is duplicated by any fragment of double-stranded (ds) DNA or dsRNA introduced into the cytoplasm of nonimmune cells. Activation is sequence-independent, is induced by ds polynucleotides as small as 25 bp in length, and is not duplicated by single-stranded polynucleotides. In addition to causing abnormal MHC expression, the ds nucleic acids increase the expression of genes necessary for antigen processing and presentation: proteasome proteins (e.g., LMP2), transporters of antigen peptides; invariant chain, HLA-DM, and the costimulatory molecule B7.1. The mechanism is different from and additive to that of γ-interferon (γIFN), i.e., ds polynucleotides increase class I much more than class II, whereas γIFN increases class II more than class I. The ds nucleic acids also induce or activate Stat1, Stat3, mitogen-activated protein kinase, NF-κB, the class II transactivator, RFX5, and the IFN regulatory factor 1 differently from γIFN. CpG residues are not responsible for this effect, and the action of the ds polynucleotides could be shown in a variety of cell types in addition to thyrocytes. We suggest that this phenomenon is a plausible mechanism that might explain how viral infection of tissues or tissue injury triggers autoimmune disease; it is potentially relevant to host immune responses induced during gene therapy.
Unlabeled cholera toxin inhibits [125I]thyrotropin binding to thyrotropin receptors on thyroid plasma membranes. Maximal inhibition by cholera toxin does not exceed 40%, whereas unalbeled thyrotropin completely inhibits [125I]thyrotropin binding to these same membranes. Kinetic analyses of the binding data are compatible with the view that the cholera toxin decreases the number of receptor sites available to thyrotropin and that the mechanism by which the cholera toxin inhibits [125I]thyrotropin binding to these receptor sites involves both competitive and noncompetitive elements.
FRTL-5 rat thyroid cells grown and maintained in a medium containing 0.05 nM thyrotropin have a 10-fold higher number of alpha 1-adrenergic receptors on their cell surface than FRTL-5 cells maintained in the absence of thyrotropin in their medium. The increased number of alpha 1-adrenergic receptors, measured as increased specific [3H]prazosin binding per microgram of DNA, is not associated with any changes in Kd values for prazosin. Thyrotropin increases the number of alpha 1-adrenergic receptors by inducing their biosynthesis, as evidence by the inhibitory effects of cycloheximide or actinomycin D; the effect on biosynthesis is cAMP-mediated, since 8-bromoadenosine 3',5'-cyclic monophosphate, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine can mimic the thyrotropin effect in both extent and time course. The alpha 1-adrenergic receptors on FRTL-5 thyroid cells have been functionally linked to iodide efflux into the follicular lumen and to the iodination of thyroglobulin--i.e., to the formation of thyroid hormones; the alpha 1-adrenergic receptor signal is mediated by Ca2+ rather than by cAMP and involves arachidonic acid intermediates. The present data thus describe a unique upregulation phenomenon wherein the sequential expression of two receptors (thyrotropin and alpha 1-adrenergic) and two distinct signal systems (cAMP and Ca2+) are apparently a necessary prelude to thyroid hormone homoeostasis.
Plasma membranes of cells from retroorbital tissue have been prepared from the Harderian glands of guinea pigs and have been characterized as being reasonably free of other subcellular structures by electron microscopy and by enzyme-marker analyses. Both bovine thyrotropin and a proteolytic derivative of bovine thyrotropin with exophthalmogenic activity but without thyroid-stimulating activity specifically bind to these membranes. Gammaglobulin from the sera of patients with malignant exophthalmos increases the binding of both pituitary factors, whereas binding is not similarly increased by gammaglobulin from the sera of individuals who are not exophthalmic. The increased binding caused by the gammaglobulin from exophthalmic patients is the same whether the sera are positive or negative for the long-acting thyroid stimulator. Present binding experiments do not indicate a direct interaction between the gammaglobulin and the plasma membranes of the cells from Harderian glands. A mechanism for the pathogenesis of human exophthalmos is proposed on the basis of these data.
A heritable connective tissue disorder of cattle, dermatosparaxis, is characterized by an extreme fragility of the skin and the presence of additional peptides at the N-terminal extremities of the collagen alpha chains, p-alpha(1) and p-alpha(2). The existence of an enzyme activity is demonstrated in normal connective tissues that is capable of cleaving these additional N-terminal peptides from dermatosparaxic collagen. The activity is demonstratable with dermatosparaxic collagen in solution, as well as with reconstituted dermatosparaxic collagen fibrils polymerized in vitro. It has a pH optimum of about 7.0 and is inhibited by EDTA and mercaptoethanol. Differences in K(m) and V(max) values exist depending on the substrate utilized, i.e., p-alpha(1) or p-alpha(2); and the presence of additional amounts of one substrate, p-alpha(1), alters the concentration requirement for the second substrate, p-alpha(2). The product of the excision reaction with p-alpha(1) as substrate is an equimolar amount of normal alpha(1) monomer; the product when p-alpha(2) is substrate is an equimolar amount of normal alpha(2) monomer. The enzyme is present in normal calf skin, tendon, aorta, cartilage, and lung; it can be demonstrated in the skin of rats and humans. The enzyme activity is absent in dermatosparaxic connective tissues, thus suggesting that dermatosparaxis is caused by the absence of a normal enzyme function rather than by the production of an abnormal collagen.
The procollagen peptidase activity of calf tendon has been purified. The enzyme has a high degree of specificity for native procollagen and converts both pro alpha1 and alpha2, to alpha1 and alpha2, respectively. The purified enzyme is an endopeptidase which excises the amino terminal peptide extensions of the precursor chains in block; the molecular size and amino-acid composition of the excised peptides compare favorably with those predicted in previous reports. Antisera to the enzyme and to procollagen have been prepared and have been used to characterize the enzyme, the enzymatically excised peptides, and the enzyme-peptide complex in reaction mixtures.
When membrane vesicles prepared from a D-lactate dehydrogenase mutant of E. coli ML 308-225 are treated with a homogeneous preparation of D-lactate dehydrogenase, the enzyme binds to the vesicles and they regain the capacity to catalyze D-lactate oxidation and D-lactate-dependent active transport. Although membranebound enzyme increases linearly with addition of increasing quantities of enzyme, reconstituted transport activity and D-lactate oxidation are saturable functions of the amount of enzyme bound. The maximal specific transport activity obtained in the reconstituted system is similar in magnitude to that of wild type vesicles. Titration studies with 2-(N-dansyl)-aminoethyl-beta-D-thiogalactoside demonstrate that there is at least a 7- to 8-fold excess of lac carrier protein relative to D-lactate dehydrogenase. Hydroxybutynoate-inactivated enzyme does not bind to the vesicles, indicating that the coenzyme moiety is critically involved in binding. Conformational changes are also apparently involved since 0.6 M guanidine.HCl is required for optimal binding and reconstitution. The relative unreactivity of reconstituted vesicles towards vinylglycolic acid suggests that D-lactate dehydrogenase is bound to the outer surface of the reconstituted vesicles.
Experimental systemic lupus erythematosus (SLE) can be induced in mice by immunization with a human monoclonal antibody to DNA that bears a common idiotype (16/6Id). These mice generate antibodies to 16/6Id, antibodies to DNA, and antibodies directed against nuclear antigens. Subsequently, manifestations of SLE develop, including leukopenia, proteinuria, and immune complex deposits in the kidney. In contrast, after immunization with 16/6Id, mice lacking major histocompatibility complex (MHC) class I molecules generated antibodies to 16/6Id but did not generate antibodies to DNA or to nuclear antigen. Furthermore, they did not develop any of the above clinical manifestations. These results reveal an unexpected function of MHC class I in the induction of autoimmune SLE.
The binding of bovine TSH (thyroid stimulating hormone), LH (luteinizing hormone), and their subunits to the TSH receptor of beef thyroid membranes was compared to stimulation by these agents of adenylate cyclase [ATP pyrophosphate-lyase(cyelizing), EC 4.6.1.1] in the same membranes, glucose oxidation in dog thyroid slices, and the secretory process in mouse thyroids in vitro (colloid droplet formation) and in vivo (hormone release). The β-subunits of TSH and LH can bind to the TSH receptor and can activate thyroid function in vitro . In contrast, the α-subunit of TSH binds negligibly to the TSH receptor and has very low potency for stimulation of thyroid function (except for colloid droplet formation). Neither binding nor the biological activity of the β-subunits can be accounted for by TSH contamination, whereas this cannot be ruled out for α-TSH. LH binds to the TSH receptor even better than the β-subunit of TSH but the increased binding does not result in a corresponding activation of thyroid function. Neither α- nor β-TSH alone can induce more than 4-8% of the response to intact TSH in any of the investigated parameters. It is proposed that the β-subunit has within its structure the primary determinants which are necessary to stimulate biological activity, whereas the α-subunit imposes conformational changes on the β-subunit which in intact TSH promote binding and biological activity commensurately but in LH promote only binding.
Hybridoma cells have been obtained by fusing P3-NS1/1-Ag4-1 mouse myeloma cells with spleen cells from mice immunized with solubilized preparations of the thyrotropin receptor. Five clones were produced that secrete a monoclonal antibody whose binding to thyroid membranes is specifically inhibited by unlabeled thyrotropin. The antibody interacts with functioning thyroid cells in culture but not with nonfunctioning cells; this interaction is prevented by thyrotropin. The antibodies are capable of competitively blocking thyrotropin binding to bovine thyroid membrane preparations; they prevent 125I-labeled thyrotropin binding to a solubilized preparation of the glycoprotein component of the bovine thyrotropin receptor but are unable to inhibit 125I-labeled thyrotropin binding to liposomes containing gangliosides at comparable concentrations. They prevent 125I-labeled thyrotropin binding to rat, bovine, or human (Graves disease) thyroid membrane preparations. They do not stimulate adenylate cyclase activity in thyroid membrane preparations but can inhibit thyrotropin-stimulated iodide uptake by functioning thyroid cells in culture.
