A subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor in systemic lupus erythematosus.

In systemic lupus erythematosus, antibodies against double-stranded DNA are a major contributor to renal disease. We have previously demonstrated that the pentapeptide Asp/Glu-Trp-Asp/Glu-Tyr-Ser/Gly is a molecular mimic of double-stranded DNA. This sequence is also present in the extracellular domain of murine and human NMDA (N-methyl-D-aspartate) receptor subunits NR2a and NR2b. Here we show that the NR2 receptor is recognized by both murine and human anti-DNA antibodies. Moreover, anti-DNA antibodies with this cross-reactivity mediate apoptotic death of neurons in vivo and in vitro. Finally, we show that the cerebrospinal fluid of a patient with systemic lupus erythematosus contains these antibodies and also mediates neuronal death via an apoptotic pathway. These observations indicate that lupus antibodies cross-react with DNA and NMDA receptors, gain access to cerebrospinal fluid and may mediate non-thrombotic and non-vasculitic abnormalities of the central nervous system.

Induction of autoreactive B cells allows priming of autoreactive T cells.

A novel mechanism for breaking T cell self tolerance is described. B cells induced to make autoantibody by immunization of mice with the non-self protein human cytochrome c can present the self protein mouse cytochrome c to autoreactive T cells in immunogenic form. This mechanism of breaking T cell self tolerance could account for the role of foreign antigens in breaking not only B cell but also T cell self tolerance, leading to sustained autoantibody production in the absence of the foreign antigen.

Simultaneous synthesis of histone and DNA in synchronously dividing Tetrahymena pyriformis.

Histone and DNA syntheses have been studied in synchronously dividing Tetrahymena pyriformis GL. During the heat treatment necessary to synchronize cultures of this amicronucleate protozoan, the DNA content of the already polyploid macronucleus increases. When the cells begin synchronous division, their DNA content is reduced in a stepwise process which is closely paralleled by reduction of macronuclear histone content. During cell division, the contents of DNA and histone decrease by slightly more than twofold, and in the subsequent S phase, DNA and histone increase simultaneously to 85% of the values expected if all chromosomes were to double. The first step in the process of reduction of DNA and histone contents is their decrease in excess of twofold, and this is accomplished by removal of extrusion bodies from the nuclei of dividing cells. The second step is a mechanism which allows, in effect, only 70% of the chromatin in the average nucleus to duplicate. Such partial duplication suggests that both histone and DNA syntheses in synchronous Tetrahymena depend upon a regulatory mechanism, the mediating elements of which are localized in only certain chromosomes.

Erythema chronicum migrans and Lyme arthritis: cryoimmunoglobulins and clinical activity of skin and joints.

We report the presence of serum cryoimmunoglobulins in patients with attacks of a newly described epidemic arthritis--Lyme arthritis--and in some patients with a characteristic skin lesion--erythema chronicum migrans--that sometimes precedes the onset of the arthritis. Seven patients who had cryoimmunoglobulins at the time of the skin lesion have developed arthritis; four patients without them have not. The cryoglobulins in patients with the skin lesion consisted primarily of immunoglobulin M (IgM); those in patients with arthritis often included both IgM and IgG. These findings support the hypothesis that a common origin exists for the skin and joint lesions and suggest that circulating immune complexes may have a pathogenetic role in Lyme arthritis.

Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus.

The RNP and Sm antigens recognized by lupus erythematosus antibodies are located on discrete particles containing single small nuclear RNA's complexed with proteins. The antigens Ro and La are also on ribonucleoproteins. The small RNA's in ribonucleoproteins with Ro are discrete, like those associated with RNP and Sm; in contrast, ribonucleoproteins with La contain a striking highly banded spectrum of small RNA's from uninfected cells as well as virus-associated RNA from adenovirus-infected cells.

The U2 small nuclear ribonucleoprotein particle as an autoantigen. Analysis with sera from patients with overlap syndromes.

We identified eight patients whose sera contained autoantibodies to the U2 small nuclear ribonucleoprotein (snRNP), an RNA protein particle involved in the splicing of newly transcribed messenger RNA. Each of these patients had an overlap syndrome that included features of either systemic lupus erythematosus (SLE), scleroderma, and/or polymyositis. We then used these sera to characterize the autoantigenic polypeptides of the U1 and U2 snRNP particles. In immunoblots, all sera contained antibodies to the B" polypeptide of the U2 snRNP. A subset of these sera that more effectively immunoprecipitated the native U2 particle contained an additional antibody system that recognized the A' polypeptide of this snRNP. Antibodies eluted from the B" protein bound the A polypeptide of the U1 snRNP and vice versa. Moreover, antibodies to the B" polypeptide were accompanied by antibodies to the 68K and C polypeptides of the U1 snRNP. Finally, the A' and B" polypeptides remained physically associated after the U2 particle was cleaved with RNase. Thus these sera contain multiple autoantibody systems that, at one level, target two physically associated antigenic polypeptides of the U2 particle and, at another, target two snRNP particles which are associated during the splicing of premessenger RNA. These linked autoantibody sets provide further evidence that intact macromolecular structures are targeted by the immune response in SLE and related diseases.

Identification of autoantibodies to RNA polymerase II. Occurrence in systemic sclerosis and association with autoantibodies to RNA polymerases I and III.

In this study, autoantibodies to RNA polymerase II from sera of patients with systemic sclerosis have been identified and characterized. These antibodies immunoprecipitated polypeptides of 220 kD (IIA) and 145 kD (IIC), the two largest subunits of RNA polymerase II, and bound both subunits in immunoblots. These polypeptides were immunoprecipitated by the anti-RNA polymerase II monoclonal antibody 8WG16, which recognizes the carboxyl-terminal domain of the 220-kD subunit, and their identity to the proteins bound by human sera was confirmed in immunodepletion studies. Sera with anti-RNA polymerase II antibodies also immunoprecipitated proteins that were consistent with components of RNA polymerases I and III. In vitro transcription experiments showed that the human antibodies were an effective inhibitor of RNA polymerase II activity. In indirect immunofluorescence studies, anti-RNA polymerase II autoantibodies stained the nucleoplasm, as expected from the known location of RNA polymerase II, and colocalized with the anti-RNA polymerase II monoclonal antibody. The human sera also stained the nucleolus, the location of RNA polymerase I. From a clinical perspective, these antibodies were found in 13 of 278 patients with systemic sclerosis, including 10 with diffuse and three with limited cutaneous disease, but were not detected in sera from patients with other connective tissue diseases and from normal controls. We conclude that anti-RNA polymerase II antibodies are specific to patients with systemic sclerosis, and that they are apparently associated with antibodies to RNA polymerases I and III. These autoantibodies may be useful diagnostically and as a probe for further studies of the biological function of RNA polymerases.

Tissue factor activity in lymphocyte cultures from normal individuals and patients with hemophilia A.

The procoagulant material of lymphocytes has been characterized as tissue factor. Lymphocytes stimulated with phytohemagglutinin or the purified protein derivative of the tubercle bacillus developed procoagulant activity with incubation in tissue culture. While this material corrected the prolonged clotting time of factor VIII (AHF) deficient plasma, we have shown, utilizing a sensitive radioimmunoassay, that no AHF antigen was present in the cell cultures. Further, we have demonstrated this material to be tissue factor by coagulation techniques and immunological cross-reactivity. The published data regarding factor VIII synthesis is reviewed in light of these observations and comments are made regarding the role of the lymphocyte procoagulant.

Circulating immune complexes in Lyme arthritis. Detection by the 125I-C1q binding, C1q solid phase, and Raji cell assays.

We have found immunoglobulin (Ig) G-containing material consistent with immune complexes in the sera of patients with Lyme arthritis. It was detected in 29 of 55 sera (55%) from 31 patients by at least one of three assays: (125)I-C1q binding, C1q solid phase, or Raji cell. The presence of reactive material correlated with clinical aspects of disease activity; it was found early in the illness, was most prominent in sera from the sickest patients, was infrequent during remissions, and often fluctuated in parallel with changes in clinical status. The results in the two C1q assays showed a strong positive correlation (P<0.001). They were each elevated in 45% of the sera and were usually concordant (85%). In contrast, the Raji cell assay was less frequently positive and often discordant with the C1q assays. In sucrose density gradients, putative circulating immune complexes sedimented near 19S; they, too, were detected best by the two assays based on C1q binding. An additional 7S component was found in some sera by the (125)I-C1q binding assay. Serum complement was often above the range of normal in patients with mild disease and normal in patients with severe disease but did not correlate significantly with levels of circulating immune complexes. IgM and IgG rheumatoid factors were not detectable. These findings support a role for immune complexes in the pathogenesis of Lyme arthritis. Their measurement, by either the (125)I-C1q binding assay or by the C1q solid phase assay, often provides a sensitive index of disease activity. Moreover, the complexes are likely sources of disease-related antigens for further study of this new disorder.

Antibodies from patients with connective tissue diseases bind specific subsets of cellular RNA-protein particles.

We characterized the RNA-containing antigens precipitated by sera from 260 patients with positive antinuclear antibodies. 49 individuals, most of whom had systemic lupus erythematosus or Sjögren's syndrome, possessed antibodies that precipitated the previously identified RNP, Sm, Ro, and La antigens either singly or in combinations. These antigens, which are located on discrete sets of small nuclear or cytoplasmic RNA-protein particles, exhibited a number of antigenic interrelationships. One patient's serum recognized a new particle containing a small RNA which we have called Th; it also precipitated the Ro complexes. Other patients with systemic lupus erythematosus, hepatitis B virus infection, juvenile rheumatoid arthritis, myositis, and rheumatoid arthritis had antibodies that precipitated specific subsets of ribosomal RNA and transfer RNA. One patient's serum contained a monoclonal immunoglobulin G that precipitated ribosomes. Most of these antibodies identified antigenic determinants constituted at least in part of protein. The specificity of the proteins bound to particular cellular RNA, probably explains the exquisite precision with which antibodies from rheumatic disease patients discriminate among RNA subsets. Such sera should be useful probes for investigating specific roles that different RNA and RNA-protein complexes play in cellular metabolism.

Autoantibodies to DNA-dependent protein kinase. Probes for the catalytic subunit.

DNA-dependent protein kinase (DNA-PK) is an important nuclear enzyme which consists of a catalytic subunit known as DNA-PKcs and a regulatory component identified as the Ku autoantigen. In the present study, we surveyed 312 patients in a search for this specificity. 10 sera immunoprecipitated a large polypeptide which exactly comigrated with DNA-PKcs in SDS-PAGE. Immunoblot analysis demonstrated that this polypeptide was recognizable by a rabbit antiserum specific for DNA-PKcs. Although the patient sera did not bind to biochemically purified DNA-PKcs in immunoblots or ELISA, they were able to deplete DNA-PK catalytic activity from extracts of HeLa cells in a dose-dependent manner. We conclude that these antibodies should be useful probes for studies which aim to define the role of DNA-PK in cells. Since six sera simultaneously contained antibodies to the Ku protein, these studies suggest that relatively intact forms of DNA-PK complex act as autoantigenic particles in selected patients.

The major core histone antigenic determinants in systemic lupus erythematosus are in the trypsin-sensitive regions.

Antibody responses against nucleosome core histones in systemic lupus erythematosus have been shown, by immunoblotting, to be directed largely against the trypsin-sensitive regions of the histones. These occur at the N-terminal regions of all 4 core histones and at the C-terminal ends of H2A and H3. Since these regions are often not the most antigenic when individual histones are used as immunogens, and appear to be exposed in the nucleosome, the active immunogens in systemic lupus erythematosus seem likely to be chromatin-bound, rather than free, histones.

The role of the epidermal growth factor receptor in microbial infections of the gastrointestinal tract.

The epidermal growth factor receptor (EGFr) is a transmembrane glycoprotein with an intrinsic tyrosine kinase. Ligand-binding to the EGFr activates cell signaling, phosphorylates protein kinases, and rearranges cytoskeletal proteins - responses that resemble those induced by microbial attachment to cell surfaces, a process known to be mediated by host cell receptors in a number of cases. This article critically reviews the possible role played by the EGFr in microbial colonization, and discusses how modulation of the EGF-EGFr axis may affect infection of the gastrointestinal tract.

Isolation of human monocytes on re-orienting gradients of Percoll.

We have developed a rapid and simple method for isolating monocytes from peripheral blood. After mononuclear cells are isolated on a Ficoll-Hypaque gradient, the monocytes are purified by sedimentation for 5 min through a re-orienting gradient of Percoll. The method is easily reproducible and yields approximately half of the monocytes in staring blood, with minimal contamination by lymphocytes. Although both monocytes and lymphocytes have similar densities, separation occurs because these cells migrate at differing rates through the gradient to reach their equilibration point. The recovered monocytes are viable and functional; over 95% of them exclude trypan blue and they adhere to glass and plastic and ingest latex particles normally. By virtue of the rapidity and gentle nature of its centrifugation steps, the method exposes cells to only very limited non-physiologic conditions. For this reason and because the monocytes are maintained in suspension, opportunities for functional alterations are minimized.

A method to detect particle-specific antibodies against Ku and the DNA-dependent protein kinase catalytic subunit in autoimmune sera.

Sera from patients with systemic lupus erythematosus, polymyositis, scleroderma, and mixed connective tissue disease are frequently characterized by the presence of high levels of autoantibodies directed against linked sets of nuclear proteins. One of these autoantigen systems is made up of Ku and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), proteins that are essential for double-strand DNA break repair and for the related process of V(D)J recombination. Ku and DNA-PKcs bind avidly to DNA ends in vivo and in vitro and form an active protein kinase complex. One hypothesis is that this assembled nucleoprotein particle, rather than its component proteins, is a primary trigger for the autoimmune response and thus a major target for the resulting autoantibodies. To screen for particle-specific antibodies, we developed an assay in which the fully native nucleoprotein particle is reconstituted in vitro and is tethered to the surface of an ELISA plate via a streptavidin-biotin linkage. These particles are recognized efficiently by monoclonal antibodies and by autoantibodies present in patient sera. The assay may detect a broader spectrum of epitopes than a conventional ELISA in which Ku and DNA-PKcs are adsorbed directly to a plastic surface. The method will be advantageous for high-throughput screening for antibodies and other ligands that bind the assembled DNA-dependent protein kinase complex.

A simple fluorescence method for surface antigen phenotyping of lymphocytes undergoing DNA fragmentation.

Apoptosis, a metabolically active process of programmed cell death characterized by DNA fragmentation, is believed to play an important role in development of lymphocyte repertoires and in embryogenesis. Studies of this phenomenon would be greatly facilitated by the development of a simple assay capable of identifying and isolating intact apoptotic cells. A rapid fluorescence assay which identifies relatively small, intact cells containing fragmented DNA is described in this report. Thymocytes in which DNA fragmentation is induced by culture with or without dexamethasone are readily identified by their bright blue fluorescence after a 15 min treatment with Hoechst 33342, a DNA binding fluorochrome which diffuses through cell membranes. Since Hoechst 33342 staining does not require destruction of the cell membrane, it is possible to directly phenotype cell surface antigen expression on Hoechst 33342bright lymphocytes by conventional immunofluorescence techniques and to evaluate membrane integrity of Hoechst 33342bright cells by dye exclusion criteria. The advantages of this system are that it: (1) is rapid and simple, (2) quantitates the percentage of cells fragmenting their DNA and presumably undergoing apoptosis, (3) permits standard immunofluorescence staining of cell surface markers to identify even minor cell subsets of presumably apoptotic cells within heterogeneous populations, (4) provides the tools (fluorescence activated cell sorting) for purifying intact cells containing fragmented DNA for further biochemical studies, and (5) provides a means for identifying cells which exclude vital dyes and in which DNA fragmentation will eventually result in cell death.

Transfer of porcine endogenous retrovirus across hollow fiber membranes: significance to a bioartificial liver.

BACKGROUND: A porcine endogenous retrovirus (PERV) capable of infecting human cells has been identified. This study was designed to determine whether hollow fiber membranes, such as those used in a bioartificial liver, block the transfer of PERV. METHODS: Three hollow fiber cartridges (HFCs) were studied in duplicate: cellulose fibers with 70 kD nominal molecular weight cut-off (MWCO), polysulfone fibers with 400 kD MWCO, and mixed cellulose fibers with 200 nm porosity. PK15 cells (porcine kidney cell line), known to produce PERV, were grown in the intraluminal compartment of HFCs fiber cartridges. Samples of medium were collected from both intraluminal and extraluminal compartments of the HFCs fiber cartridge during 14 days of culture. Samples were screened for PERV using reverse transcription polymerase chain reaction. All positive samples were tested for PERV infectivity in human 293 cells. RESULTS: PERV was detected in all samples from the intraluminal space and all intraluminal samples seemed to infect 293 cells. All extraluminal samples from the fibers of 200 nm porosity tested positive for PERV. Detection of PERV in the extraluminal space was delayed by fibers of 400 kD MWCO and 70 kD MWCO until at least day 3 and day 7, respectively, after inoculation of PK15 cells. Positive extraluminal samples from fibers of 400 kD MWCO and 70 kD MWCO did not infect 293 cells. CONCLUSION: Pore size, membrane composition, and duration of exposure influenced the transfer of PERV across HFCs. Some HFCs decrease the risk of viral exposure to patients during bioartificial liver therapy.

Patterns of autoimmunity to nucleoproteins in patients with systemic lupus erythematosus.

The ANAs described above can be accounted for on the basis of an immune response to just three nucleoprotein structures--the nucleosome, the U1 snRNP, and the Ro particle. When these nucleoproteins are looked at in turn, the following picture emerges. The nucleosome is identified by both anti-histone and anti-DNA antibodies. Anti-histone H1 and anti-histone H2B antibodies predominate and tend to occur together. They, as well as the anti-DNA antibodies with which they appear to be linked, recognize external features of the intact nucleosome. The U1 snRNP is recognized by both anti-U1 RNP and anti-Sm antibodies. Most so-called anti-U1 RNP antisera actually contain several linked sets of different antibodies that are directed against various polypeptides (68K, A, and C) found on the U1 snRNP. Anti-Sm antibodies are linked to the occurrence of anti-U1 RNP antibodies. The Ro particle is recognized by both anti-La and anti-Ro antibodies, and almost all sera that contain anti-La antibodies also contain anti-Ro antibodies. Thus, it appears that these three nucleoprotein particles become direct focal points for autoimmune responses in SLE. It is difficult to explain such focused responses on the basis of a general defect in immune regulation or spontaneous B-lymphocyte hyperactivity. Rather it appears that these nucleoproteins themselves are directly involved in determining which B-lymphocyte clones become activated. Thus, the simplest rationalization for the patterns with which these autoantibodies occur is to invoke the possibility that the particles themselves are directly triggering autoimmune responses.

Cytoprotective influence of ZVAD-fmk and glycine on gel-entrapped rat hepatocytes in a bioartificial liver.

BACKGROUND: This study was designed to determine if an anti-necrotic compound, glycine, and/or an anti-apoptotic agent, ZVAD-fmk, improved the viability and function of hepatocytes in a bioartificial liver. METHODS: Isolated rat hepatocytes were entrapped in collagen gel (1.0-10.0 x 10(6) cells/mL) and cultured in serum-free medium (1:10 ratio of gel:media) supplemented with glycine alone, ZVAD-fmk alone, or glycine and ZVAD-fmk. The cytoprotective effects of glycine and ZVAD-fmk on gel-entrapped rat hepatocytes (GERH) were determined after anoxic exposure (0-20 hours). Cell functionality (measured by urea production), cell viability (quantitated by vital staining with fluorescein diacetate:ethidium bromide [FDA:EB]), and the mechanism of cell death (verified by electron microscopy and DNA fragmentation studies) were determined for each condition. RESULTS: The viability of GERH declined gradually and then stabilized 12 hours after hepatocyte isolation. The rate of urea production by GERH was directly proportional to the number of viable hepatocytes. Apoptotic death predominated at low cell density, and necrotic cell death became significant at high cell density. Hepatocyte necrosis became more significant after exposure to longer periods of anoxia (4, 8, 12, and 20 hours). ZVAD-fmk provided dose-dependent cytoprotection to GERH with an optimum benefit at a concentration of 60 mumol/L. After anoxic exposure or under high cell density culture, glycine demonstrated a maximum benefit of inhibiting necrosis at a concentration of 3 mmol/L. The beneficial effects of glycine and ZVAD-fmk were additive. CONCLUSIONS: The metabolic activity of a hepatocyte bioartificial liver may benefit from the use of cytoprotective agents such as ZVAD-fmk and glycine.

The effect of TGF alpha on intestinal solute transport.

The effect of transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) on 3-O-methylglucose transport was examined in vitro under short-circuited conditions in stripped rabbit jejunum. Mucosal EGF, 60 ng/ml, stimulated a significant increase in net 3-O-methylglucose transport (Jnet 0.67 +/- 0.15 vs. 0.90 +/- 0.15 microEq/cm2/h; P less than 0.03; n = 6) due to an increased mucosal to serosal flux (Jms 1.2 +/- 0.2 vs. 1.5 +/- 0.2 microEq/cm2/h; P less than 0.03). In contrast, TGF alpha, when applied to both mucosal and serosal surfaces at concentrations of either 60 (n = 6) or 150 (n = 9) ng/ml had no effect on either mucosal to serosal (Jms) or net transport (Jnet) of 3-O-methylglucose. TGF alpha did induce a significant increase in the serosal to mucosal flux (Jsm 60 ng/ml 0.44 +/- 0.02 vs. 0.51 +/- 0.03, 150 ng/ml 0.55 +/- 0.03 vs. 0.64 +/- 0.05 microEq/cm2/h; P less than 0.05). When brush border surface area was examined after exposure to either 60 ng/ml TGF alpha or saline vehicle for 2 h in in vivo isolated jejunal loops no significant difference was found (control 53 +/- 1.9; n = 35 vs. TGF alpha 52 +/- 1.9 microns 2; n = 29). Bioactivity of transforming growth factor alpha was assessed by an gastric acid secretion bioassay and found to be intact. These data provide further evidence for separate and distinct functional roles for these peptides in some biological systems.