A subpopulation of WIL-2 cell calreticulin molecules is associated with RO/SS-A ribonucleoprotein particles.

A subpopulation of human calreticulin (CR) molecules that is reactive with human Ro/SS-A autoimmune sera was identified in a nucleic acid- enriched Wil-2 cell fraction derived by anion exchange column chromatography. Further resolution of this fraction by gel filtration size separation demonstrated that the appearance of CR (true mol. weight 46 kD) coincided with the emergence of Ro/SS-A ribonucleoprotein (mol. weight > 250 kD) antigenic activity and increasing 260 nm ultraviolet absorbance. This high nucleic acid fraction could be further partitioned into four small RNA-containing Ro/SS-A antigenic subfractions by a second passage over the anion exchange column. CR was enriched in one subfraction and present in the other three subfractions as well. No CR was found in the RNA-free fraction of the repartition eluate. These results represent the first direct demonstration that CR, a high-affinity calcium binding protein, exists in a form that is directly associated with all four varieties of native, human Ro/SS-A ribonucleoprotein particles (hY1-5).

Studies on the expression and immunogenicity of the B50 melanoma antigen and its relationship with calreticulin.

B50 is a 50 kDa protein antigen originally identified and isolated from cultured B16 murine melanoma cells; it is found in close association with a melanoma-specific antigen termed B700. Using a specific rabbit antiserum, B50 (or B50 cross-reactive molecules) has been shown to be expressed by 35 out of 36 cell lines, including melanomas, sarcomas, fibrosarcomas, carcinomas, gliomas, immortalized and primary fibroblasts, melanocyte and keratinocyte cell lines obtained from murine, human, hamster, swine, and canine donors. B50 expression is localized on the cellular membrane and in the cytoplasm in varying amounts in seven of the nine cell lines tested. Mice immunized to B50 demonstrated a significant tumour rejection response when subsequently challenged with B16 F10 melanoma cells. Previous studies had indicated that B50 has significant N-terminal amino acid sequence homology with calreticulin. Calreticulin, a calcium-binding protein, is part of the Ro/SS-A complex. This complex is the primary autoantigenic determinant of the autoimmune diseases systemic lupus erythematosus and primary Sjogren's syndrome. We now show that sera from patients with those diseases contain antibodies which bind B50, although B50 itself does not bind calcium. Thus, B50 and calreticulin are closely related but distinct antigens.

Impact of ultraviolet irradiation on expression of SSA/Ro autoantigenic polypeptides in transformed human epidermal keratinocytes.

SSA/Ro autoantibodies are frequently found in various autoimmune disorders including subacute cutaneous and neonatal lupus erythematosus. SSA/Ro patient sera precipitate a ribonucleoprotein complex consisting of multiple polypeptides and small RNA molecules (hY RNA). Such sera react in Western blot with at least four antigenically distinct proteins having molecular weights of 52-60 kD. Several laboratories have reported increased binding of anti-SSA/Ro patient serum to viable cultured human epidermal keratinocytes following UVB irradiation. However, it is currently unknown which SSA/Ro molecule(s) might be responsible for this increased antibody binding to UVB irradiated keratinocytes. To address this question, we studied the effect of UVB irradiation on the expression of three different polypeptide components of the SSA/Roautoantigen complex (60 kD SSA/Ro, 52 kD SSA/Ro, and 46 kD SSA/Ro (calreticulin) in A431 cells, a transformed human epidermal keratinocytes cell line. Total cellular and cell surface expression of each SSA/Ro antigenic polypeptide was examined by a whole cell ELISA and FACS using rabbit anti-synthetic peptide antisera as probes. Our results suggest that both total cellular and cell surface calreticulin, but not the 60 and 52 kD SSA/Ro polypeptides, is increased after 100 J/M2 of UVB irradiation, indicating that perturbed calreticulin expression may be primarily responsible for the UVB-induced increased binding of anti-SSA/Ro to keratinocytes. These results suggest that calreticulin could be a critical component of the SSA/Ro ribonucleoprotein complex that is involved in the pathogenesis of anti-SSA/Ro-associated photosensitive LE skin lesions.

Calreticulin is released from activated neutrophils and binds to C1q and mannan-binding protein.

The Ca2+ storage protein calreticulin is associated with the endoplasmic reticulum and shares a high degree of amino acid homology with the surface receptor C1q-R. In this study, flow cytometric analysis detected calreticulin on the neutrophil surface, which decreased during stimulation probably as a consequence of shedding, as calreticulin was found by ELISA in the cell supernatants of stimulated cells. Antibodies raised against C1q-R and calreticulin demonstrated a high degree of immunological cross-reactivity for purified calreticulin as determined by dot blot analysis. Western blots of neutrophil subcellular fractions located calreticulin in both the cytosol and cell membrane fractions; C1q-R was largely confined to the cell membrane. Calreticulin and C1q-R both bind to C1q and mannan-binding protein. Therefore, calreticulin may be shed on cell activation and may be associated with the cell membrane, where it can potentially interact with C1q and serum lectins. The implications of this are discussed.

Impact of ultraviolet radiation on the cellular expression of Ro/SS-A-autoantigenic polypeptides.

Modulation of Ro/SS-A autoantigens in epidermal keratinocytes has been implicated in the pathogenesis of photosensitive forms of Ro/SS-A-antibody-associated cutaneous lupus erythematosus (LE) such as subacute cutaneous LE and neonatal LE. Since Ro/SS-A ribonucleoprotein particles have recently been shown to be very complex molecular structures, we have performed studies to determine whether the expression of three of the Ro/SS-A antigenic polypeptides might be differentially regulated in transformed human epidermal keratinocytes following UVB radiation. Our findings indicate that both total cellular and cell surface levels of calreticulin are upregulated by UVB exposure more so than are either the 52- or 60-kD Ro/SS-A antigens. These results suggest that calreticulin could be a critical component of the Ro/SS-A ribonucleoprotein complex involved in the pathogenesis of Ro/SS-A-antibody-associated LE skin lesions.

Serological cross-reactivity between a human Ro/SS-A autoantigen (calreticulin) and the lambda Ral-1 antigen of Onchocerca volvulus.

We have cloned and sequenced a 46-kD Ro/SS-A autoantigen gene that is the human homologue of the calcium-binding protein, calreticulin. The sequence of this 46-kD Ro/SS-A protein (calreticulin) has significant homology to lambda Ral-1, a recombinant cDNA clone corresponding to a major antigen of the nematode, Onchocerca volvulus, the infectious agent of onchocerciasis. We therefore sought to determine whether antibodies produced by onchocerciasis patients might crossreact with the human 46-kD Ro/SS-A autoantigen (calreticulin). 20 of 22 sera from Liberian onchocerciasis patients who had no known evidence of autoimmune disease were found to contain antibodies that reacted with the 46-kD Ro/SS-A (calreticulin) by immunoblot analysis. Characteristic of sera reactive with Ro/SS-A antigens, some onchocerciasis sera also immunoprecipitated the Ro/SS-A-associated hY RNAs. In addition, a monoclonal antibody raised against O. volvulus organisms reacted to purified human WiL-2 cell 46 kD Ro/SS-A antigen (calreticulin) by ELISA. These results strongly suggest that onchocerciasis patients produce antibodies that crossreact with the 46-kD human Ro/SS-A autoantigen (calreticulin) and raise the possibility that infectious organisms such as O. volvulus might play a triggering or exacerbating role in the human Ro/SS-A autoimmune response.

Molecular characterization of the Ro/SS-A autoimmune response.

Human Ro/SS-A (Ro) antibodies (anti-Ro) are frequently seen in the circulation of patients with several subsets of photosensitive cutaneous lupus erythematosus (LE) and related disorders. Experimental observations have suggested that anti-Ro reacting with Ro antigens that are expressed abberantly within the epidermis might play a causal role in LE-specific skin disorders such as subacute cutaneous LE (SCLE) and neonatal LE. In this article, we will review the progress that has recently been achieved toward gaining a better understanding of the molecular configuration, genetic regulation, and function of the Ro ribonucleoprotein (RNP) antigenic complex as well as the autoimmune response with which it is associated. Work in this area has recently revealed the existence of multiple Ro autoantigen-bearing polypeptides including a 46-kd Ro protein that is virtually identical to calreticulin, a highly conserved, calcium-binding protein that is normally associated with the endoplasmic reticulum. The implications of these observations with respect to the pathogenesis of anti-Ro-associated LE skin lesions are explored.

Correlation between dermal interstitial immunoglobulin G and hypergammaglobulinemia.

The diffuse dermal immunofluorescence (DDIF) observed in human skin biopsies that is produced by fluorochrome-conjugated antisera reactive with human immunoglobulin G (IgG) has commonly been viewed in the past as an artifact of direct immunofluorescence microscopy. However, it has been our belief that DDIF, which has been observed in 14% of the biopsies processed in our laboratory, might represent an in vivo phenomenon of excess unbound dermal interstitial immunoglobulin G reflective of elevated serum IgG levels. To examine this hypothesis, we carried out a blinded prospective study of the frequency of DDIF in two groups of age- and sex-matched patients that differed with respect to the presence or absence of serum hypergammaglobulinemia (gamma globulin greater than 2.5 mg/100 ml). The subjective intensity of DDIF and the fluorescein-isothiocyanate (FITC) conjugated anti-human IgG antibody titer at which point DDIF disappeared were both compared with serum gamma globulin levels. Also, the amount of IgG that could be extracted from biopsy specimens by an overnight phosphate-buffered saline (PBS) elution procedure was quantified by solid-phase immunoassay. We noted a trend of increased DDIF intensity with increasing serum gamma globulin levels. In addition, there was a relatively weak but significant positive correlation between DDIF endpoint titers and serum gamma globulin levels (p = 0.05, r = 0.429). The strongest positive statistical correlation observed in the study was between the quantities of IgG that could be eluted from skin biopsies and serum gamma globulin levels (p = 0.01, r = 0.599). These findings suggest that DDIF is a true biologic phenomenon reflective of elevated serum gamma globulin levels and demonstrate that simple overnight elution of unbound dermal IgG can unmask the presence of disease-related, specifically-bound IgG.

IgG subclasses in the serum and skin in subacute cutaneous lupus erythematosus and neonatal lupus erythematosus.

IgG subclasses differ in their biologic and chemical properties, such as complement fixation, protein and cellular binding, and placental transfer. In this study, IgG subclasses of anti-Ro/SSA antibodies in subacute cutaneous lupus (SCLE) and neonatal lupus (NLE) are examined in the serum and in the skin. IgG subclasses in NLE beginning in utero (NLE-heart disease) are compared to subclasses in NLE beginning after birth (NLE-skin disease). Human skin was grafted onto athymic mice, mice were injected with one of eight anti-Ro/SSA maternal NLE sera (four heart block, four skin disease) or seven anti-Ro/SSA SCLE sera, and grafts were examined for IgG subclasses using monoclonal anti-human IgG subclass reagents in an immunofluorescent technique. Lesional skin was examined from four SCLE patients. IgG1 was the only IgG subclass detected in the grafts and skin lesions. IgG1 was the predominant anti-Ro/SSA IgG subclass detected in SCLE and NLE sera in an ELISA using a synthetic Ro/SSA polypeptide. These studies show that the maternal anti-Ro/SSA autoantibodies in NLE-heart disease sera are predominantly IgG1 and are therefore likely to be present in the fetus at the time of gestation, when heart block usually develops. Second, differences in the clinical presentations of NLE (in utero vs. postnatal disease) cannot be attributed to differences in anti-Ro/SSA IgG subclasses. Finally, the subclass bound in the skin in SCLE is IgG1, a subclass capable of mediating tissue injury via complement or cellular effectors.

A human Ro/SS-A autoantigen is the homologue of calreticulin and is highly homologous with onchocercal RAL-1 antigen and an aplysia "memory molecule".

The Ro/SS-A (Ro) autoantigens consist of at least four immunologically distinct proteins which are recognized by autoantibodies typically found in sera from patients with primary Sjogren's syndrome and in subsets of patients with lupus erythematosus. We recently isolated a 1.9-kb human cDNA clone which encodes one of these Ro autoantigens. Synthetic oligonucleotides corresponding to the human Ro sequence were used to amplify the homologous gene from a murine B cell cDNA library using the polymerase chain reaction. The mouse cDNA-encoded amino acid sequence was found to be 94% homologous to the human Ro sequence and is 100% homologous to murine calreticulin, a high affinity calcium-binding protein which resides in the endoplasmic and sarcoplasmic reticulum. The amino acid sequence of rabbit calreticulin is 92% homologous to both murine calreticulin and human Ro. Onchocerca volvulus and Drosophila melanogaster also have molecules that are highly homologous to human Ro. In addition, human Ro has a molecular mass, isoelectric point, and significant amino acid sequence similar to the Aplysia californica snail neuronal protein 407. These homologies suggest that this Ro protein has a very basic cellular function(s) which may in part involve calcium binding.

Molecular cloning, expression, and chromosome 19 localization of a human Ro/SS-A autoantigen.

Ro/SS-A antibodies are found in a number of human autoimmune disorders including Sjogren's syndrome and several systemic lupus erythematosus-related disorders. These heterogeneous autoantibodies are known to recognize several distinct cellular antigens. With synthetic oligonucleotides corresponding to amino acid sequence information we have isolated a full-length cDNA clone which encodes a human Ro ribonucleoprotein autoantigen. The 1,890-base pair clone contains an open reading frame that encodes a 417-amino acid, 48-kD polypeptide that migrates aberrantly at 60 kD by SDS-PAGE. Rabbit antibodies raised against this protein's recently described amino-terminal epitope react with a previously identified 52-kD human Ro protein and immunoprecipitate the human cytoplasmic RNAs. Ultraviolet light cross-linking studies suggest that this Ro protein binds each of the four major human cytoplasmic RNAs. The deduced amino acid sequence is 63% homologous to an Onchocerca volvulus antigen. Southern filter hybridization analysis indicates that this gene is not highly polymorphic and exists as a single copy in the human genome. Chromosomal localization studies place this gene on the short arm of chromosome 19 near the gene encoding the low density lipoprotein receptor.

Epidermolysis bullosa acquisita preceding the development of systemic lupus erythematosus.

In most cases of epidermolysis bullosa acquisita that occur in patients with systemic lupus erythematosus, the diagnosis of systemic lupus erythematosus is made before the development of blistering. We observed three patients with well-documented epidermolysis bullosa acquisita that developed several years before the onset of systemic lupus erythematosus. One patient was producing anti-U1RNP autoantibodies at the time epidermolysis bullosa acquisita was diagnosed, and all five produced this antibody during the systemic lupus erythematosus phase of their illness. In addition, in all five cases of epidermolysis bullosa acquisita with systemic lupus erythematosus antibodies to double-stranded DNA ultimately developed, and severe systemic lupus erythematosus and lupus nephritis developed in four patients. Sera from 15 other patients with epidermolysis bullosa acquisita without overt systemic lupus erythematosus were analyzed for systemic lupus erythematosus-related autoantibodies. Four patients were found to have at least one such autoantibody. These findings further document an association between epidermolysis bullosa acquisita and systemic lupus erythematosus and suggest that patients with systemic lupus erythematosus who present with epidermolysis bullosa acquisita may represent a subset of lupus erythematosus that puts the patient at increased risk for the development of more severe systemic illness. Patients presenting with epidermolysis bullosa acquisita, especially those who are black or Hispanic, should be monitored for the development of potentially life-threatening systemic lupus erythematosus.

A major autoepitope is present on the amino terminus of a human SS-A/Ro polypeptide.

A human SS-A/Ro antigen is present on the polypeptide component of a particle composed of hyRNA and a 60 kD protein. We have now purified the Wil-2 cell 60 kilo dalton (kD) SS-A/Ro protein and determined its amino-terminal amino acid sequence. A synthetic peptide corresponding to residues 7 to 24 of this sequence (RoSP7-24) exhibited enzyme-linked immunosorbent assay (ELISA) binding activity with immunodiffusion-defined, monospecific anti-SS-A/Ro sera. In addition, ELISA binding of monospecific anti-SS-A/Ro sera to native SS-A/Ro antigen was partially inhibited (35%) by KLH-RoSP7-24. Sera from patients known to frequently produce precipitating anti-SS-A/Ro antibody (subacute cutaneous lupus erythematosus [SCLE], 56 patients; Sjögren's syndrome [SS], 41 patients; mothers of infants with neonatal LE [NLE], 10 individuals; infants with congenital heart block [CHB], 5 patients) were tested for reactivity to RoSP7-24 in ELISA. Overall, 38% of SCLE sera, 36% of SS sera, 50% of maternal NLE sera and 20% of CHB infant sera had anti-RoSP7-24 binding levels greater than 2 standard deviations above the mean of that of normal individuals. Of the sera which had anti-SS-A/Ro detected by double immunodiffusion and/or counterimmunoelectrophoresis, 68% of SCLE patients, 71% of SS patients, 55% of NLE mothers and 20% of CHB infants had significantly elevated RoSP7-24 ELISA binding levels. These findings strongly suggest that a major autoepitope of native human SS-A/Ro resides on the amino terminal portion of the Wil-2 SS-A/Ro 60 kD polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Ro/SS-A and the pathogenic significance of its antibodies.

Ro/SS-A is a soluble cellular protein to which antibodies are frequently directed in patients with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE)-related disorders. The pathogenic significance of these antibodies is uncertain. Our effort to further characterize the immunological and structural nature of Ro/SS-A suggests that the native human Ro/SS-A molecule is not glycosylated and consists of two disulfide linked domains. The amino terminus of the smaller domain contains a major epitope reactive with anti-Ro/SS-A sera. We have isolated and characterized a complementary DNA (cDNA) clone which encodes the Ro/SS-A polypeptide. The Ro/SS-A gene does not appear to be highly polymorphic and exists as a single copy on the short arm of chromosome 19. The isolation of the Ro/SS-A cDNA should allow for a more complete characterization of Ro/SS-A epitopes which should help further elucidate the importance of Ro/SS-A antibodies in the pathogenesis of disease.

Molecular characterization of human Ro/SS-A antigen. Amino terminal sequence of the protein moiety of human Ro/SS-A antigen and immunological activity of a corresponding synthetic peptide.

The Ro/SS-A antigen was purified from an Epstein-Barr virus-transformed human B lymphoblastoid cell line. The amino terminal amino acid sequence of the 60-kD polypeptide bearing this antigenic epitope was determined to be: (formula; see text) A peptide composed of residue 6-19 was synthesized by the solid-phase method. Immunodiffusion-defined monospecific autoimmune sera to Ro/SS-A reacted with this synthetic peptide in ELISA, whereas autoantibodies with other specificities such as anti-La/SS-B and anti-Sm, as well as normal human sera, were not reactive. In addition, rabbit anti-peptide 6-19 antisera reacted specifically with native human Ro/SS-A antigen in ELISA. Furthermore, this synthetic peptide inhibited the binding of rabbit anti-peptide antiserum to native human Ro/SS-A. An additional synthetic peptide corresponding to residues 7-24 partially inhibited the binding of a patient anti-Ro/SS-A serum to native Ro/SS-A. These results suggest that the amino terminal portion of the molecule represents a major epitope of Ro/SS-A. The determination of the amino acid sequence of Ro/SS-A and the availability of synthetic peptide(s) bearing this antigen should provide additional approaches to further characterize the autoimmune response to this antigen.

Immunoglobulin class and subclass profile of the Ro/SS-A autoantibody response.

We have developed an enzyme-linked immunosorbent assay (ELISA) for autoantibody to Ro/SS-A antigen (anti-Ro/SS-A) in order to more fully characterize the autoimmune response that occurs to this antigen in patients with subacute cutaneous lupus erythematosus (SCLE). The microtiter plate-immobilized, biochemically purified Ro/SS-A antigen reacted with anti-Ro/SS-A antibody, but not with other closely related specificities (anti-La/SS-B, anti-SM, anti-U1-RNP) or normal sera. The optimal pH of antigen-antibody reaction in this ELISA was 7.2. The binding of sera containing anti-Ro/SS-A was inhibited 80% by preincubation with the same amount of Ro/SS-A antigen used for coating the plate. Although 11 of the 14 (79%) SCLE sera studied had precipitating anti-Ro/SS-A antibody by immunodiffusion, 13 (93%) sera had abnormally elevated IgG, IgA, or IgM ELISA binding levels. A good correlation between IgG anti-Ro/SS-A ELISA binding levels and immunodiffusion titers was observed (r - 0.8588, p less than or equal to 0.001) suggesting that IgG is the major anti-Ro/SS-A antibody class detected by double immunodiffusion, Sera with a combination of high rheumatoid factor levels (latex 3+ or higher) and high anti-Ro/SS-A titers (1:8 or higher in immunodiffusion) tended to give an abnormally high IgM anti-Ro/SS-A ELISA binding levels. After rheumatoid factor activity was removed by absorption with heat-aggregated human IgG, a 50% decrease in IgM anti-Ro/SS-A ELISA binding was noted. On the other hand, absorption of rheumatoid factor-negative sera that contained high IgM anti-Ro/SS-A binding activity did not significantly decrease ELISA binding levels. Prednisone and 6-azathioprine reduced the level of IgG anti-Ro/SS-A autoantibody in sera of treated SCLE patients by 50%. The IgG subclass profile of anti-Ro/SS-A autoantibody was analyzed by using mouse monoclonal antibodies specific for the 4 human IgG subclasses. Of anti-Ro/SS-A positive SCLE sera, 91% had predominantly IgG1 subclass autoantibody. The coexistence of IgM and IgG anti-Ro/SS-A autoantibody and the predominance of the IgG1 subclass is compatible with the possibility that this autoantibody response is under T-cell control. The predominance of IgG1 in the autoimmune response to Ro/SS-A antigen in SCLE patients is consistent with the hypothesis that antibody dependent cell mediated cytotoxicity could be an important immunologic effector mechanism in this disorder.

Relationship between circulating anti-Ro/SS-A antibody levels and skin disease activity in subacute cutaneous lupus erythematosus.

Anti-Ro/SS-A antibody levels in 80 serum specimens from 12 patients with subacute cutaneous lupus erythematosus (SCLE) were determined by immunodiffusion and enzyme-linked immunosorbent assay in order to examine the changes in this autoantibody response with time and to study the relationship between levels of this antibody and SCLE skin disease activity. Anti-Ro/SS-A antibody levels were found to vary considerably over time in a given patient when measured by both assays. Several patients who did not have detectable levels of this antibody at the time of their initial examination were found to be antibody positive at follow-up examinations. However, no significant relationship was found between antibody levels in either assay and SCLE skin disease activity. While not ruling out a pathogenetic role for this antibody in the elicitation of SCLE skin lesions, these results would suggest that other factors are likely to be involved.

Identification of the SS-A/Ro intracellular antigen with autoimmune sera.

Autoantibodies to SS-A/Ro antigen have been described in the sera of certain patients with Sjögren's syndrome, systemic lupus erythematosus and in neonates with lupus and/or congenital heart block. SS-A/Ro is a trypsin-sensitive intracellular antigen which appears to be associated with another intracellular antigen SS-B/La in tissue extracts. This study describes a simple method of separation of SS-A/Ro and SS-B/La antigenic activity by differential salt elution in polybuffer ion exchange chromatography. The partially purified SS-A/Ro antigen was separated by polyacrylamide gel electrophoresis under non-denaturing conditions and identified by Western blot analysis as a single polypeptide of 61,000 Da. The pI of SS-A/Ro antigen was 4.67. The partially purified SS-A/Ro antigen derived from gel electrophoresis could be adapted to an enzyme linked immunosorbent assay (ELISA) for the detection of antibody.