Self-reactive repertoire of tight skin (TSK/+) mouse: immunochemical and molecular characterization of anti-cellular autoantibodies.

The tight skin (TSK/+) mouse has been proposed as an experimental model for progressive systemic sclerosis because of the biochemical alterations in collagen synthesis and pathological similarities to the human disease. Here, we report the analysis of tight skin mice sera for the presence of anti-cytoplasmic and anti-nuclear autoantibodies and determination of the frequency of hybridomas producing anti-cellular autoantibodies. The binding specificity of TSK mAbs to nuclear and cytoplasmic antigens such as keratin, actin, vimentin, and mitochondria was determined. Of 71 monoclonal antibodies that we have studied, only 3 appear to bind to foreign as well as self-antigens, indicating that the majority of these antibodies do not belong to the class of natural autoantibodies. Our results also showed that the frequency of hybridomas producing anti-nuclear and anti-cytoplasmic antibodies was higher in TSK mice than in C57BL/6 pa/pa, the control mouse strain, used in these studies. The results of the analysis of V gene usage showed that the majority of anti-cytoplasmic and anti-nuclear antibodies are encoded by genes from a restricted number of VH and VK genes families. In the sera of TSK mice we have detected the presence autoantibodies specific for cytoplasmic antigens in addition to anti-nuclear and anti-topoisomerase I antibodies which are characteristic of scleroderma. Since the presence of anti-cytoplasmic antibodies has not been described in scleroderma, the significance of their production in tight skin mice is not clear. However, the presence of such autoantibodies in the animal model provides a basis for investigation of this type of antibodies in human disease.

High-specificity in-situ hybridization. Methods and application.

We describe a technique of in-situ hybridization using oligonucleotide probes employing the expression of immunoglobulin VH genes as a model. Optimal conditions for hybridization with the 35S-labeled oligonucleotide probes were established with monoclonal B-cell lines that express VH genes of known nucleic acid sequence. The range of sensitivity and specificity achieved with this technique is documented. Under conditions of high stringency, this method can detect the expression of highly related VH hypervariable regions.

Independent analysis of the 16/6 idiotype lupus model. A role for an environmental factor?

The recent description of a lupus-like disease in normal mice after injections of human mAb that bind DNA and carry the common Id 16/6 Id has excited much attention. In an effort to reproduce this model we have performed two experiments using eight human mAb three of which bear the 16/6 Id. Despite using an injection protocol very similar to that of the original authors and waiting for up to 1 yr we were unable to detect any autoantibodies or any evidence of renal disease. We suspect that our failure to reproduce the model may be due to differences in either or both the batches of CFA used, or in the animal house environments. It supports the view that a mosaic of effects is required to induce clinical expression of an autoimmune disease.

Molecular analysis of a germ line-encoded idiotypic marker of pathogenic human lupus autoantibodies.

Id-16/6 is an idiotypic marker found in both IgM and IgG antibodies, as well as in the tissue lesions of patients with SLE. The prototypic Id-16/6+ mAb is 18/2, whose VH3-derived H chain is encoded by an unmutated germ-line gene. We found that the H chains of VH3-derived Id-16/6+ antibodies contain the major determinants of Id-16/6. Moreover, B cell clones from which those antibodies were harvested produce RNA that hybridized under conditions of high stringency to oligonucleotide probes corresponding to the CDR of the VH segment of 18/2. Western blots of Id-16/6+ mAbs with anti-Id confirmed the association of the Id with H chains. Id-16/6 can identify a subgroup of VH3-derived antibodies we have termed the 18/2 CDR family. However, Id-16/6 can also be expressed in some antibodies unrelated to the 18/2 CDR family. No characteristic Ag-binding specificity was found among the members of the 18/2 CDR family. The principal phenotypic feature shared by all known members of the family is Id-16/6.

An immunoglobulin light chain from a lupus-prone mouse induces autoantibodies in normal mice.

Autoantibodies against the 70-kD U1 RNP nucleoprotein autoantigen and DNA were elicited in normal BALB/c mice with a purified Ig light chain. This light chain, derived from a lupus-prone MRL-lpr/lpr mouse, has two distinctive properties: it contains an idiotypic marker recognized by a monoclonal MRL-lpr/lpr anti-snRNP autoantibody, and the amino acid sequence of its third hypervariable region (CDR3) is homologous to a sequence in an antigenic region of the 70-kD U1 RNP polypeptide. The results demonstrate that an Ig idiotype that mimics an autoantigen can induce autoimmunization.

Polyspecificity of human monoclonal antibodies reactive with Mycobacterium leprae, mitochondria, ssDNA, cytoskeletal proteins, and the acetylcholine receptor.

The origin of autoantibodies against ubiquitous autoantigens (e.g., single-stranded (SS) DNA, cytoskeletal proteins, mitochondria) is obscure. Patients with lepromatous leprosy have many such autoantibodies in their serum. In order to study the polyspecificities of human autoantibodies expressed during infection with Mycobacterium leprae we prepared human monoclonal antibodies derived from the fusion of peripheral blood lymphocytes of a patient with lepromatous leprosy to the human lymphoblastoid line GM 4672. Hybridomas were tested for binding to a DNAse-treated sonicate of M. leprae and a panel of autoantigens. Of the primary (uncloned) cultures, 14% bound ssDNA, 35% bound M. leprae, 11% bound both M. leprae and ssDNA, and 16% bound to mitochondria. Several also bound to the acetylcholine receptor of Torpedo marmorata. Monoclonal antibodies derived from separate primary cultures revealed similar cross-reactions between several autoantigens and M. leprae. In addition, one antibody was identified which bound to mitochondria and the acetylcholine receptor, and which was recognized by an anti-idiotypic antibody which bears the "internal image" of the acetylcholine receptor. These results suggest that antigenic mimicry may play a role in eliciting autoantibody expression from the immune repertoire.

Malignant granular lymphoproliferation after Epstein-Barr virus infection: partial immunologic reconstitution with interleukin-2.

This report describes a patient who developed a malignant proliferation of granular lymphocytes following Epstein-Barr virus (EBV) infection. For many months, his illness resembled prolonged infectious mononucleosis with persistent fatigue, fever, leukocytosis, and serologic evidence of recent primary EBV infection. After approximately 1 year, however, he developed progressive granular lymphocytosis and extensive lymphocytic infiltration of the bone marrow and liver. Tests for EBV DNA in pre- and postmortem tissue samples using a sensitive DNA hybridization technique were negative. Southern blot analysis of DNA prepared from blood mononuclear cells demonstrated clonal T-cell antigen receptor gene rearrangement. Despite increased numbers of circulating lymphocytes with the morphology and surface phenotype of normal donor natural killer (NK) cells, the patient's NK activity was consistently depressed in a standard in vitro assay. However, in vitro incubation with interleukin-2 (IL-2), but not with alpha- or gamma-interferon, increased the NK activity of the patient's lymphocytes. Intravenous recombinant IL-2 treatment transiently increased the patient's blood NK activity and was associated with seroconversion to EBV nuclear antigens but failed to affect the progression of his disease. Our findings indicate that clonal granular lymphocytic proliferation may develop after EBV infection and confirm the utility of DNA hybridization analysis in distinguishing monoclonal from benign immunoreactive lymphoproliferation. Furthermore, our results suggest that certain functionally inert neoplastic granular lymphocytes acquire NK activity when exposed to IL-2.

A single germline VH gene segment of normal A/J mice encodes autoantibodies characteristic of systemic lupus erythematosus.

These experiments tested the hypothesis that unmutated germline genes from normal mice can encode autoantibodies. We found that the unmutated VHIdCR gene segment, which encodes a large proportion of antiarsonate antibodies in A/J mice, also encodes antibodies with the ability to bind to DNA and cytoskeletal proteins. After Ars immunization, at a time when the VHIdCR gene segment mutates and antibody affinity for the hapten increases, reactivity with the autoantigens was lost. Six antibodies obtained after immunization with Ars bound both the Ars and DNA. Results of competitive inhibition assays suggested that the same variable region site in the antibodies bound to both Ars and DNA. The properties of the individual germline-encoded antibodies, which include reactivity to both DNA and cytoskeletal proteins, suggest that autoantibodies characteristic of SLE might be a subset of antibodies encoded by unmutated germline V genes.

Gaucher's disease and chronic lymphocytic leukemia. Possible pathogenetic link between Gaucher's disease and B-cell proliferations?

The authors report a case of Gaucher's disease with chronic lymphocytic leukemia. The diagnosis of Gaucher's disease was confirmed by electron microscopy and glucocerebrosidase assay. There may be a pathogenetic link between Gaucher's disease and B-cell proliferation.

Binding of cytoskeletal proteins by monoclonal anti-DNA lupus autoantibodies.

Monoclonal anti-DNA antibodies produced by hybridomas derived from MRL-lpr/lpr mice and human lupus patients were found to bind to the cytoskeleton of mink lung cells. When tested by indirect immunofluorescence, 17/29 human monoclonal anti-DNA antibodies reacted with the cytoskeleton; 4 of the 29 also produce antinuclear reactions with epithelial cells. The cytoskeletal staining was not inhibited by prior treatment of the cells with DNase, but it was completely blocked by prior incubation of the monoclonal antibodies with DNA and other nucleic acids. The ability of the polynucleotides to inhibit the cytoskeletal staining corresponded to their ability to bind to the antibodies in competitive immunoassays. An (Fab')2 preparation of a monoclonal antibody bound to the cytoskeleton as well as the whole immunoglobulin. The effect of colcemid on the staining pattern, the blocking effect of a monoclonal antivimentin antibody, and results with nitrocellulose blots of cellular proteins indicated that the cytoskeletal protein to which the antibodies bound was vimentin.

Expression of multiple isozymes of granulocyte, monocyte, and macrophage esterases in polycythemic Friend erythroleukemia cells.

We examined the expression of cytochemical markers of myeloid and monocyte-macrophage differentiation in conjunction with ultrastructural studies of different malignant erythroleukemic cells isolated from mice infected with the Friend polycythemic virus complex (FLV-P). The amounts of fluoride-sensitive and resistant nonspecific esterase activity increased with the progression of malignancy. Isoelectric focusing resolved this enzyme activity into 13 isozymes in the most malignant Friend cell type tested. These same isozymes were found in the adherent cell population of normal spleens. Two of these isozymes were shown to have chloroacetate esterase activity characteristic of granulocytes. Despite these myeloid and monocyte characteristics, light and electron microscopy showed no morphological evidence of differentiation in either of these lineages. This study demonstrates that the Friend erythroleukemic cell contains markers of three different hemopoietic cell types. The expression of myeloid, monocytic, and erythroid traits in these erythroleukemic cells can be used to monitor their malignant progression.

Polyspecificity of monoclonal lupus autoantibodies produced by human-human hybridomas.

We studied the serologic properties of monoclonal autoantibodies that were produced by hybridomas derived from lymphocytes of patients with systemic lupus erythematosus. The hybridomas were made by fusion of a human lymphoblastoid cell line, GM 4672 (derived from a patient with multiple myeloma), with peripheral-blood or splenic lymphocytes from six patients with lupus. Thirty monoclonal autoantibodies, selected for their ability to react with denatured DNA, were analyzed. Eighteen of them reacted with three or more additional polynucleotides, including native DNA, left-handed double-helical DNA (Z-DNA), poly(l), and poly(dT). Ten reacted both with nucleic acids and the phospholipid cardiolipin. The multiple binding reactions of the monoclonal autoantibodies may be explained by the presence of appropriately spaced phosphodiester groups in both the polynucleotides and the phospholipid. The sharing of antigenic groups by polymers of different natures may contribute to the apparent diversity of serologic reactions in systemic lupus erythematosus. These findings suggest that DNA itself need not be the immunogenic stimulus for autoantibody formation in this disease.

Expression of a chronic granulomatous disease-like defect by fluoride-exhausted neutrophils.

Neutrophils incubated with 20 mM F- express a respiratory burst without degranulating or performing phagocytosis. After 60 min of F- treatment, the burst is exhausted and cannot be restarted. Neutrophils so treated have a microbicidal defect similar to that of chronic granulomatous disease (CGD): they kill Str. mitis at a nearly normal rate, but show a marked impairment in the destruction of S. aureus. They differ from CGD neutrophils in that they also display a defect in motility. This defect, however, is not so severe as to seriously impair their ability to kill bacteria by mechanisms that are independent of endogenously generated microbicidal oxidants.

Peripheral blood myeloid progenitor cell cultures in patients with hypereosinophilic syndrome (CFU-eos in hypereosinophilic syndrome).

Myeloid progenitor cell cultures (CFU-C) were established in a double-layer agar system with peripheral blood mononuclear cells from 13 patients with the hypereosinophilic syndrome (HES). Normal controls produced 49% +/- 3.5% eosinophil colonies; results in 7 of the 13 HES patients were within the normal range, while in 5, the proportion of eosinophil colonies was greater than 3 standard deviations above the normal mean, and in 1 patient there was a low proportion of eosinophil colonies. The production of an increased proportion of eosinophil colonies correlated with more aggressive disease. Experiments in which normal progenitor cells were cultured over feeder layers of mononuclear cells demonstrated that cells of 3 of the 5 patients had an excess production of eosinophil colony-stimulating activity. When HES patients progenitor cells were cultured over normal feeder layers, 2 of the 5 patient samples continued to produce an increased proportion of eosinophil colonies, suggesting that these patients have an excess proportion of progenitor cells committed to eosinophil differentiation. Thus, the results demonstrated heterogeneity of growth characteristics for the HES patients. None, however, had the colony growth characteristic of acute or chronic myelogenous leukemia.

In vitro culture of circulating CFUEOS from normal donors.

Using an accurate technique for staining and scoring soft agar cultures, this study defines the incidence of circulating CFUEOS in normal donors. With whole mononuclear cells as the source of colony stimulating factor (CSF) and fetal calf serum, 49.6% +/- 3.5 of the colonies were eosinophilic; with human placental conditioned media and fetal calf serum 33.2% +/- 12.9 of the colonies were eosinophilic, with AB serum and whole mononuclear cells in the feeder layer 58.6% +/- 9.1 of the colonies were eosinophilic. The percentage of mixed neutrophil/eosinophil colonies was similar under varying culture conditions suggesting the presence of circulating progenitor cells capable of producing both lines.

Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.

An inherited abnormality of neutrophil adhesion. Its genetic transmission and its association with a missing protein.

Neutrophils from a five-year-old boy with recurrent bacterial infections failed to spread on surfaces, leading to a severe defect in chemotaxis and a mild impairment in phagocytosis. Failure to spread was also seen in a fraction of the neutrophils from the patient's mother and sister, but cells from his father and brother were normal. Gel electrophoresis revealed that a protein with a molecular weight of 110,000 daltons (designated gp 110) present in the particulate fraction of normal neutrophils was absent from the patient's cells, and that its levels were below normal in cells from his mother and sister but normal in neutrophils from his father and brother. These findings suggest that gp 110 is necessary for the spreading of neutrophils onto surfaces, that the functional abnormality in the patient's cells is caused by its absence, and that deficiency of gp 110 is an X-linked congenital disease.

Termination of the respiratory burst in human neutrophils.

Recent evidence has suggested that a particulate O(2) (-)-forming system is responsible for the respiratory burst in activated neutrophils. The respiratory burst is normally a transient event, lasting only 30-60 min. To investigate the mechanism by which the burst is terminated, we examined the O(2) (-)-forming activity of neutrophil particles as a function of time in the presence and absence of agents known to affect the function of intact cells. Measurements of the O(2) (-)-forming capacity of the particles against time of exposure of neutrophils to opsonized zymosan, a potent stimulating agent, revealed a rapid fall in activity when exposure was continued beyond 3 min. Exposure to zymosan under conditions in which the myeloperoxidase system was inactive (i.e., in the presence of myeloperoxidase inhibitors, or in the absence of oxygen) resulted in a substantial increase in the initial O(2) (-)-forming activity of particles from the zymosan-treated cells, but did not prevent the sharp fall in activity seen when zymosan exposure exceeded 10 min. The fall in activity was, however, prevented when activation took place in the presence of cytochalasin B (1.5 mug/ml), an agent thought to act largely by paralyzing the neutrophil through an interaction with its microfilament network.We conclude from these findings that the termination of the respiratory burst results at least in part from the inactivation of the particulate O(2) (-)-forming system. This inactivation involves at least two processes which probably act simultaneously. One is the destruction of the system through the action of myeloperoxidase. The other appears to require active cell motility and is independent of oxygen. The current view holds that the O(2) (-)-forming system of the neutrophil is located in the plasma membrane. It may be that the second process involves the internalization and degradation of this membrane-bound system.

Genetic studies of autoimmunity and retrovirus expression in crosses of New Zealand black mice I. Xenotropic virus.

The relationship between expression of xenotropic virus and the development of autoimmunization was studied in the progeny of crosses between New Zealand Black (NZB) and SWR mice. The (F1 X SWR) and F2 progeny segregated into three phenotypes: high-virus, low-virus, and virus-negative; F1 and (F1 X NZB) progeny were always high-virus. Autoantibodies, immune deposit nephritis and lymphomas developed in the progeny of these crosses. The virological phenotype of the animal could be dissociated from the presence of either autoantibodies or nephritis. For example, mice that expressed titers of virus as high as the NZB parent failed to develop signs of autoimmunization, even up to 24 mo of age. By contrast, some (F1 X SWR) and F2 mice that expressed low titers of virus developed autoimmune disease. Furthermore, a proportion of virus-negative mice produced autoantibodies and were found to have typical immune deposit nephritis. No viral antigens could be detected in the renal lesions of such virus-negative animals. By contrast with the dissociation between expression of virus and occurrence of nephritis, the presence of antibodies to DNA correlated with the development of renal lesions. We conclude that the genes that determine the expression of infectious xenotropic virus in NZB mice segregate independently from those that are involved in the autoimmune disease of these animals.

Treatment of lupus nephritis in adult (NZB + NZW)F1 mice by cortisone-facilitated tolerance to nucleic acid antigens.

Adult female (NZB + NZW)F1 mice were treated with cortisone, cortisone with tolerogen (isologous NZB IgG-nucleosides conjugates) or cortisone with isologous IgG free of nucleosides. Other treatments also included tolerogen or isologous IgG alone, and cortisone together with denatured DNA. All untreated mice died by 10 mo of age. Cortisone prolonged the survival rate. This effect was further improved by combined treatment of cortisone and tolerogen. Prolonged survival was accompanied by a decrease in proteinuria. Other treatments failed to influence either survival or proteinuria. Although cortisone did not prevent the appearance of antibody to denatured DNA, cortisone and tolerogen suppressed them in most of the animals. Preexisting antibody to denatured DNA was reduced by cortisone and cortisone and tolerogen, but not by cortisone and IgG. In contrast, antibody to native DNA bore no relationship to therapy. Animals living beyond 1 yr of age, regardless of the treatment, fall into three histopathological categories: (a) severe nephritis, as in untreated animals, (b) moderate nephritis (with absence of severe alteration of the glomerular basement membrane, i.e. the histological counterpart of prolonged survival), (c) minimal nephritis. In a small number of animals treated with cortisone or cortisone and IgG and in 6/20 animals treated with cortisone and tolerogen, minimal lesions as judged by light, fluorescent, and electron microscopy were found. These last mice were in good health at 15-16 mo of age, twice the life-span of untreated mice. In conclusion, these data suggest that tolerance to nucleic acid antigens facilitated by cortisone offers a promising new approach to treat established murine lupus nephritis.