Autoantibodies to major and minor nuclear lamins are not restricted to autoimmune diseases.

Autoantibodies to lamins, the major polypeptide components of the nuclear lamina, have been reported in selected sera from patients with autoimmune diseases, including anti-lamin B in systemic lupus erythematosus (SLE) and anti-lamins AC in autoimmune chronic active hepatitis (CAH). We have studied the frequency, specificity, and isotypy of autoantibodies to major and minor lamins by immunoblotting on purified rat liver lamins in 190 sera from normal controls (n = 62), rheumatic disease controls (n = 42), and autoimmune disease patients (n = 86). The frequency of anti-lamin in normal controls was 85.5%, and ranged from 77 to 100% in the other groups. Anti-lamin frequency was not related to age, sex, or disease duration. Reactivity with lamin A or with minor lamins only was observed with 7 various sera and 2 normal sera, respectively. Between groups, the proportions of reactive sera were not different for lamins AC (18-47%) and for lamin B (22-36%). In particular, anti-lamin B and anti-lamins AC were not more common in SLE or CAH than in normal sera. The most frequent lamin specificity of SLE sera was anti-lamins ABC. Anti-lamin isotypes were IgG and/or IgM. Titers of IgM antibodies were not higher in any group. However, IgG anti-lamin titers were higher in CAH than in normal, ankylosing spondylitis, or SLE sera. The highest end point titers (greater than or equal to 1:3200) were observed with CAH, SLE, and rheumatoid arthritis (RA) sera with IgG anti-lamins AC, B, or ABC, or with IgM anti-lamins ABC. None of these SLE and RA patients had evidence of liver disease. Reactivity with minor lamins was more frequent in CAH. We conclude that anti-lamin autoantibodies are present in sera from most individuals and that the highest titers are found in sera from patients with autoimmune diseases.     

Human autoantibodies to lamin B receptor are also anti-idiotypic to certain anti-lamin B antibodies.

Autoantibodies reactive with nuclear envelope proteins are mainly detected in human sera from patients with liver diseases. Some of these antibodies are directed to lamin B, lamins A and C, or to the lamin B receptor (LBR). We show here that the latter one are anti-idiotypic to certain anti-lamin B antibodies. Using an enzyme-linked immunosorbent assay specific for lamins we found that serum M containing anti-LBR antibodies inhibited the binding to lamins of anti-lamin B autoantibodies from three of five sera tested. Similar results were obtained using patient's M purified IgG. The binding of monoclonal IgM, lambda anti-lamin B antibodies produced by a lymphoblastoid cell line derived from the patient's blood lymphocytes was also inhibited. Absorption of serum M with nuclei abolished the inhibitory activity. No inhibition was recorded with normal sera or sera containing other antinuclear specificities. Anti-LBR antibodies did not alter the binding to lamins of sera containing anti-lamins A and C antibodies. Altogether these findings demonstrate that anti-LBR antibodies are also combining site related anti-idiotypic antibodies (Ab2) to certain anti-lamin B antibodies, provide further evidence for discrete specificities among anti-lamin B antibodies and suggest that the occurrence of autoantibodies to nuclear envelope antigens may be under idiotypic regulation.     

Onset and regulation of anti-lamin B autoantibody production is independent of the level of polyclonal activation

Anti-lamin B autoantibodies are associated with both systemic lupus erythematosus (SLE) and autoimmune liver disease. We examined the possibility that the underlying clinical feature in patients with anti-lamin B autoantibodies might be chronic autoimmune liver disease, and whether the hypergammaglobulinemia present in both disorders is involved in generating anti-lamin B autoantibodies. A lamin B fusion protein (MLB1), consisting of amino acids 77-533 of lamin B fused to TrpE, was used to screen sera from 84 patients with SLE for anti-lamin B autoantibodies. 3/4 prototype human lamin B antisera, 5/84 SLE sera (6%), and 0/30 sera from healthy individuals reacted with MLB1 on immunoblots at a 1:500 dilution. Of the 9 anti-lamin B autoantibody positive patients studied, all but 1 fulfilled at least four ARA criteria for SLE. None of the patients displayed evidence of chronic autoimmune liver disease, suggesting that autoimmune liver disease is not strongly associated with anti-lamin B antibodies in SLE. In SLE, as in "lupoid hepatitis", anti-lamin B autoantibodies are often produced transiently during periods of increased disease activity. Although polyclonal hypergammaglobulinemia is also associated with increased activity of both diseases, anti-lamin B autoantibody production in 2 patients was independent of total immunoglobulin levels, antibodies to irrelevant proteins, and production of some other autoantibodies. Thus, polyclonal activation is insufficient to explain either the initiation or regulation of anti-lamin B autoantibody production, supporting the hypothesis that antinuclear antibodies are antigen-selective.     

Antibodies to nuclear lamins in autoimmune liver disease

Antibodies to nuclear lamins were detected in sera of patients with autoimmune liver disease. In indirect immunofluorescence tests, these sera revealed staining of the nuclear periphery. Using isolated nuclei, nuclear matrices, nuclear lamina-pore complexes, and chromatographically purified lamins as antigen source, the nuclear lamins A, B, and C were identified as reactive antigens in immunoblotting experiments. The lamins were also identified by 2-D gel electrophoresis. Antibodies to nuclear lamins occurred in 12 of 16 cases of active lupoid hepatitis, but not in 35 patients with the disease in remission. However, only 3 of 37 sera of patients with primary biliary cirrhosis contained anti-lamin antibodies. Autoimmune liver disease sera reacted preferentially with lamins A/C and less frequently with lamin B or lamins A/B/C.     

Lamins A and C are present in the nuclei of early porcine embryos,with lamin A being distributed in large intranuclear foci

Gametogenesis and embryogenesis are dynamic developmental stages marked by extensive modifications in the organization of the genome and nuclear architecture. In the literature it is conveyed that only B-type lamins are required in these early stages of development and that A-type lamins are not present or required until differentiation of specific cell types associated with specialized tissue is initiated. To assess the presence of nuclear structures that are putatively involved in genome regulation, we investigated the distribution of lamin proteins throughout the early stages of porcine embryonic development, using testes tissue sections, oocytes and in-vitro fertilized (IVF) porcine embryos and employing anti-lamin antibodies. We have shown that anti-lamin A staining is present at the one-cell, two-cell, four-cell, and six- to eight-cell stages of early porcine embryo development, but diminishes at the morulae and blastocyst stages. Large intranuclear anti-lamin A foci are prominent in the early preimplantation stages. Both anti-lamin A/C and anti-lamin B staining were clearly present in all embryonic stages. Immature porcine oocytes revealed lamin rings using the monoclonal anti-lamin A/C antibody and many immature oocytes exhibited a pale rim staining pattern with anti-lamin A antibody. A-type lamins were not observed in sperm precursor cells. Thus, we have shown that A-type lamins and B-type lamins are present at the nuclear envelope in very early porcine embryos and that lamin A is also found in large intranuclear aggregates in two-cell to eight-cell embryos but is lacking from later embryonic stages. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Characterization of two human monoclonal IgM antibodies that recognize nuclear lamins.

Using immunofluorescence and immunoblotting techniques, we have identified monoclonal IgM lambda from two patients that are specific for lamins A and C and lamin B, respectively. Lamins A, B, and C are peripheral membrane proteins of the nuclear envelope with structural similarities to cytoplasmic intermediate filament proteins. When studied by indirect immunofluorescence on rat tissues, the serum containing anti-lamin B IgM stained smooth and striated muscles in addition to nuclear envelopes. Lamin B antibodies affinity purified from this serum were able to label muscle cells, suggesting that lamin B shares an epitope(s) with an unidentified muscular component(s). Since in an enzyme-linked immunosorbent assay there was no reactivity with a panel of proteins which are frequent targets of "natural" antibodies, these monoclonal IgM appear to belong to the rare category of IgM that possess a restricted specificity.     

Antibodies to nuclear lamin proteins in liver disease

Sera of patients with autoimmune liver disease contained antibodies reactive with nuclear lamins. These antigens were identified in immunoblotting experiments, using isolated nuclei, nuclear matrices, nuclear lamina-pore complexes and purified lamins as antigen source. The lamins were, furthermore, characterized by 2-dimensional gel electrophoresis. Antibodies to nuclear lamins were found in 75 per cent of the active lupoid hepatitis cases, but not in patients with inactive disease. Anti-lamin antibodies were detected in 8 per cent of primary biliary cirrhosis sera. The autoimmune liver disease sera recognized predominantly the nuclear lamins A/C, and less frequently the lamins A/B/C or lamin B.     

Antibodies to nuclear Lamin proteins in liver Disease

Sera of patients with autoimmune liver disease contained antibodies reactive with nuclear lamins. These antigens were identified in immunoblotting experiments, using isolated nuclei, nuclear matrices, nuclear lamina-pore complexes and purified lamins as antigen source. The lamins were, furthermore, characterized by 2-dimensional gel electrophoresis. Antibodies to nuclear lamins were found in 75 per cent of the active lupoid hepatitis cases, but not in patients with inactive disease. Anti-lamin antibodies were detected in 8 per cent of primary biliary cirrhosis sera. The autoimmune liver disease sera recognized predominantly the nuclear lamins A/C, and less frequently the lamins A/B/C or lamin B.     

Cell cycle changes in A-type lamin associations detected in human dermal fibroblasts using monoclonal antibodies

A new panel of anti-A-type lamin monoclonal antibodies was generated. Epitope mapping was performed by immunoblotting against GST—lamin fusion peptides. Epitopes were mapped to four different regions of human lamin A and three different regions of human lamin C. The distribution of A-type lamins was compared with the distribution of the proliferation marker Ki67 in proliferating and quiescent cultures of human dermal fibroblasts (HDFs) using a double indirect immunofluorescence assay. Antibodies that had been mapped to a region of the lamin C tail stained the nuclear envelope of proliferating and quiescent cells equally brightly. In contrast, antibodies recognizing epitopes in the head domain and rod domain of lamins A and C and the tail domain of lamin A stained the nuclear envelope of quiescent cells strongly but reacted poorly or not at all with the nuclear envelope of proliferating cells. Changes in the level of expression of lamins A and C were not detected in immunoblotting assays. However, epitope masking was revealed, and this occurred by two distinct mechanisms. Epitope masking in the head domain of lamins A and C occurred as a result of protein phosphorylation. Epitope masking in the rod domain of lamins A and C and in the tail domain of lamin A occurred through a physical association between the lamin and chromatin and/or other nuclear proteins. The cell cycle timing of epitope masking was investigated in HDFs that had been restimulated after serum starvation. Extensive epitope masking in restimulated cells only occurred after cells had passed through mitosis. These results are consistent with the hypothesis that rearrangement of A-type lamin filaments, as cells progress from a quiescent to a proliferating state, results in altered lamina associations.This revised version was published online in November 2005 with corrections to the Cover Date.     

Herpes simplex virus 2 UL13 protein kinase disrupts nuclear lamins

Herpesviruses must cross the inner nuclear membrane and underlying lamina to exit the nucleus. HSV-1 US3 and PKC can phosphorylate lamins and induce their dispersion but do not elicit all of the phosphorylated lamin species produced during infection. UL13 is a serine threonine protein kinase conserved among many herpesviruses. HSV-1 UL13 phosphorylates US3 and thereby controls UL31 and UL34 nuclear rim localization, indicating a role in nuclear egress. Here, we report that HSV-2 UL13 alone induced conformational changes in lamins A and C and redistributed lamin B1 from the nuclear rim to intranuclear granular structures. HSV-2 UL13 directly phosphorylated lamins A, C, and B1 in vitro, and the lamin A1 tail domain. HSV-2 infection recapitulated the lamin alterations seen upon expression of UL13 alone, and other alterations were also observed, indicating that additional viral and/or cellular proteins cooperate with UL13 to alter lamins during HSV-2 infection to allow nuclear egress.     

Modulation of protein composition of nuclear lamina. Reduction of lamins B1 and B2 in livers of griseofulvin-treated mice.

Chronic griseofulvin (GF) intoxication of mice leads to severe alterations of the hepatocytic intermediate filament cytoskeleton similar to that found in alcoholic hepatitis in humans (i.e., derangement and diminution of the keratin filament network and appearance of cytoplasmic aggregates of keratin-containing material, termed Mallory bodies). To investigate eventual alterations of nuclear lamins under these pathologic situations monoclonal antibodies were produced. One of these, GL-35, was directed to lamin B1 and lamin B2. Immunofluorescence microscopy revealed in GF-treated livers, in comparison to normal mouse livers, a highly reduced immunoreaction for lamins B1 and B2, whereas the staining for lamins A and C was unchanged. This reduction of both B-type lamins occurred before detectable alterations of keratin filaments and was reversible after cessation of GF intoxication. A diminished content of both B-type lamins in relation to lamins A and C in GF-intoxicated livers was also revealed by analysis of isolated nuclear envelopes on two-dimensional gels. Moreover, there was a clear predominance of more acidic isoelectric variants of lamins B1 and B2. In contrast to the reduced amount of B-type lamin proteins no reduction in the concentration of the mRNA for lamin B1 was found. For lamin B2 even an increase of mRNA was detected in GF-treated livers. These results indicate that GF not only interferes with expression of the keratin intermediate filament skeleton of hepatocytes but also leads to selective alterations of the nuclear lamins, most likely by posttranslational modifications of intermediate filament proteins.     

Lupus autoantibodies discriminate between the highly homologous Sm polypeptides B/B' and SmN by binding an epitope restricted to B/B'.

The ubiquitous Sm polypeptides B/B' (28 and 29 kD) and the highly homologous tissue-specific Sm N polypeptide (29 kD) share several autoepitopes recognized by systemic lupus erythematosus (SLE) sera. Previous studies on the antigenicity of nuclear antigens recognized by human autoantibodies have not discriminated between ubiquitous and tissue-specific forms. We set out to examine whether a tissue-specific nuclear antigen, Sm N, is autoantigenic in SLE by comparing the immunoreactivity of the most unique sequences in this polypeptide. Synthetic peptides from the two regions of least sequence homology that occur between Sm N and Sm B/B', a dodecamer (amino acid residues 179-190 containing five substitutions) and an undecamer (residues 203-213 containing four substitutions) were coupled to a carrier protein. These conjugates were used to quantify IgG anti-peptide antibodies in sera from patients with SLE. Of 43 sera with anti-Sm specificity, six bound to the B/B' 179-190 peptide but not to the N version. None of 17 anti-Sm-negative SLE sera bound these peptides. The second region of least sequence homology between N and B/B' (203-213) was not antigenic. Our data suggest that a subset of SLE patients with anti-Sm reactivity have IgG autoantibodies capable of discriminating between Sm N and SmB/B' polypeptides by binding a previously unreported SmB/B'-specific autoepitope. The data also indicate that brain and heart-specific anti-Sm antibodies do not exist in SLE sera, suggesting that these tissues do not participate in the induction or maintenance of the autoimmune anti-Sm response.     

The truncated prelamin A in Hutchinson-Gilford progeria syndrome alters segregation of A-type and B-type lamin homopolymers

Hutchinson-Gilford progeria syndrome (HGPS) is a dominant autosomal premature aging syndrome caused by the expression of a truncated prelamin A designated progerin (Pgn). A-type and B-type lamins are intermediate filament proteins that polymerize to form the nuclear lamina network apposed to the inner nuclear membrane of vertebrate somatic cells. It is not known if in vivo both type of lamins assemble independently or co-assemble. The blebbing and disorganization of the nuclear envelope and adjacent heterochromatin in cells from patients with HGPS is a hallmark of the disease, and the ex vivo reversal of this phenotype is considered important for the development of therapeutic strategies. Here, we investigated the alterations in the lamina structure that may underlie the disorganization caused in nuclei by Pgn expression. We studied the polymerization of enhanced green fluorescent protein- and red fluorescent protein-tagged wild-type and mutated lamins in the nuclear envelope of living cells by measuring fluorescence resonance energy transfer (FRET) that occurs between the two fluorophores when tagged lamins interact. Using time domain fluorescence lifetime imaging microscopy that allows a quantitative analysis of FRET signals, we show that wild-type lamins A and B1 polymerize in distinct homopolymers that further interact in the lamina. In contrast, expressed Pgn co-assembles with lamin B1 and lamin A to form a mixed heteropolymer in which A-type and B-type lamin segregation is lost. We propose that such structural lamina alterations may be part of the primary mechanisms leading to HGPS, possibly by impairing functions specific for each lamin type such as nuclear membrane biogenesis, signal transduction, nuclear compartmentalization and gene regulation.     

Sera from patients with rheumatic diseases recognize different epitope regions on the 52-kD Ro/SS-A protein.

Patients suffering from systemic lupus erythematosus (SLE) or Sjögren's syndrome (SS) often contain autoantibodies directed to the Ro(SS-A) complex. In this study the antigenic determinants on two of the components of the Ro complex, i.e. the Ro60 and the Ro52 polypeptides, were investigated. Anti-Ro+ sera were selected by counter-immunoelectrophoresis. Depending on the detection method, 59-68% of the SLE patients produced anti-Ro but not anti-La antibody, while 72-81% of the SS patients produced both anti-Ro and anti-La antibody. Immunoprecipitation of recombinant Ro-proteins showed that 61 sera (87%) were reactive with both Ro proteins, seven sera with Ro60 only, one serum with Ro52 only, and one serum did not precipitate the proteins at all. The anti-Ro60 reactivity of human sera is strongly associated with the native form of Ro60, suggesting that conformational autoepitopes are an important feature of Ro60. In the case of Ro52, frequently the residues located between amino acids 216 and 292 were essential for reactivity with the antibodies. With 70% of the lupus sera tested this appeared to be the only region important for reactivity. The antibodies of SS patients generally recognized multiple B cell epitopes located between amino acids 55 and 292. The results of this study indicate that the antigenic determinants on Ro52 are different for autoantibodies produced by lupus patients compared with those of SS patients.     

IDENTIFICATION OF THE ANTIGEN RECOGNIZED BY THE MONOCLONAL ANTIBODY BU31 AS LAMINS A AND C

The murine monoclonal antibody BU31 binds to the nuclear membrane of many cell types. The expression of the BU31 antigen has previously been shown to have an inverse correlation with the proliferative index in lung tumours, defined by Ki67 staining. The distribution of BU31-positive cells is now shown to parallel the distribution of non-dividing cells in a range of normal human and rat tissues, although neuroendocrine cells and germ cells in the testis show no reactivity. Cells grown in culture and induced to undergo growth arrest show a higher level of labelling with BU31 than their proliferating counterparts. Confocal laser scanning microscopy reveals that the BU31 antigen is distributed predominantly along the nuclear lamina, with occasional internal foci. This distribution is very similar to that of the nuclear membrane proteins lamin A and lamin C, suggesting that the BU31 antigen and lamins A and C could be one and the same. Immunoblotting using recombinant lamin proteins confirmed this proposal. Moreover, a monoclonal antibody to the non-proliferation-associated antigen, statin, also recognizes lamins A and C. These data indicate that the demonstration of lamins A and C can be used to provide information on the proliferative activity of normal and neoplastic tissues. These data also suggest a role for nuclear lamins A and C during cellular quiescence, possibly through the reorganization and maintenance of nuclear structure, or more directly through interactions with the retinoblastoma gene product or related proteins.     

The nuclear lamina: molecular organization and interaction with chromatin

The nuclear lamina is located between the inner nuclear membrane and the peripheral chromatin. It is composed mainly of nuclear lamins and lamina-associated proteins. The nuclear lamina is involved in nuclear organization, cell cycle regulation, and differentiation. As such, impairment in its architecture and/or function leads to genetic diseases and apoptosis. This article describes the molecular organization of the nuclear lamins, their assembly into filaments, their distribution within the nucleus, and the complex network of interactions between them and other proteins of the inner nuclear membrane. Recent findings unraveled evidence for specific interactions between proteins of the nuclear lamina and the chromatin. These include interactions between nuclear lamins and core histones, Lamina Associated Polypeptide 2 (LAP2), and the Barrier to Autointegration Factor (BAF) and interactions between lamin B receptor (LBR) and the chromodomain protein HP1. Taken together, these studies attribute a role for both the nuclear lamins and the lamina-associated proteins, LAP2 and LBR, in nuclear organization and nuclear assembly.     

Lamins and lamin-binding proteins in functional chromatin organization

Lamins are the major components of the nuclear lamina, a two-dimensional filamentous network at the periphery of the nucleus in higher eukaryotes, directly underlying the inner nuclear membrane. Several integral proteins of the inner nuclear membrane bind to lamins and may link the nuclear membrane to the core lamina network. The lamins and the lamin-binding proteins lamina-associated polypeptide (LAP)2beta and lamin B receptor (LBR) have been described to bind to DNA or to interact with chromatin via histones, BAF-1, and HP1 chromodomain proteins, respectively, and may provide anchorage sites for chromatin fibers at the nuclear periphery. In addition, lamin A structures on intranuclear filaments, or lamin B in replication foci have been described in the nuclear interior, but their specific roles remain unclear. An isoform of the LAP2 protein family, LAP2alpha, has been found to colocalize with A-type lamins in the nucleoplasm and might be involved in intranuclear structure organization. In the course of cell-cycle-dependent dynamics of the nucleus in higher eukaryotes, lamins as well as lamin-binding proteins seem to possess important functions during various steps of post-mitotic nuclear reassembly, including cross-linking of chromatides, nuclear membrane targeting, nuclear lamina assembly, and the formation of a replication-competent nucleus.     

Antibodies from patients with systemic lupus erythematosus and drug-induced lupus bind determinants on histone 5 (H5).

The antigenic domains of histone 5 (H5), a highly conserved variant of histone 1 (H1), were studied in relation to their reactivity with autoantibodies found in the sera of patients with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL). While some H5 antibodies cross-react with H1, adsorption and immunoblotting studies have identified H5-specific antibodies as well. After proteolytic cleavage of H5 peptides, the reactivity of sera from these patients was tested by Western immunoblotting. All SLE (9/9) and DIL (7/7) sera bound an antigenic determinant in the carboxyl (C) terminus of H5 while none of the sera bound to the amino (N) terminus or the central hydrophobic domain. Although the reactivity of DIL sera with the purified H5 peptides was weaker than that of SLE sera, the antigenic domains bound by both groups of sera were the same. These observations demonstrate that the H5 domains reacting with DIL sera are restricted to the carboxyl terminus and are therefore no less restricted than those reacting with SLE sera. Further, the potential epitopes in the carboxyl terminus of H5 do not have a high degree of sequence identity with known mammalian peptides.