Acute generalized exanthematous pustulosis: European expert consensus for diagnosis and management.

Acute generalized exanthematous pustulosis (AGEP) is a rare, usually drug-induced, acute pustular rash. Despite the lack of strong data supporting the effectiveness of topical or systemic corticosteroids in this drug reaction, they are widely used. More generally, there is no consensus on the diagnostic modalities and the management of patients with AGEP. We aimed to provide European expert recommendations for the diagnosis and management or patients with AGEP. Members of the ToxiTEN group of the European Reference Network (ERN)-skin, all dermatologists and/or allergologists with expertise in drug reactions, elaborated these recommendations based on their own experience and on a review of the literature. Recommendations were separated into the following categories: professionals involved, assessment of the diagnosis of AGEP, management of the patient and allergological work-up after the acute phase. Consensus was obtained among experts for the list of professionals involved for the diagnosis and management of AGEP, including the minimum diagnostic work-up, the setting of management, the treatments, the modalities and the timing of allergological work-up and follow-up. European experts in drug allergies propose herein consensus on the diagnosis and management of patients with AGEP. A multidisciplinary approach is warranted, including dermatologists, allergologists and pharmacovigilance services.

Supportive care in the acute phase of Stevens-Johnson syndrome and toxic epidermal necrolysis: an international, multidisciplinary Delphi-based consensus.

BACKGROUND: Supportive care is the cornerstone of management of adult and paediatric Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). However, consensus on the modalities of supportive care is lacking. OBJECTIVES: Our aim in this international multicentric Delphi exercise was to establish a multidisciplinary expert consensus to standardize recommendations regarding supportive care in the acute phase of SJS/TEN. METHODS: Participants were sent a survey via the online tool SurveyMonkey, consisting of 103 statements organized into 11 topics: multidisciplinary team composition, suspect drug management, infection prevention, fluid resuscitation and prevention of hypothermia, nutritional support, pain and psychological distress management, management of acute respiratory failure, local skincare, ophthalmological management, management of other mucosa, and additional measures. Participants evaluated the level of appropriateness of each statement on a scale of 1 (extremely inappropriate) to 9 (extremely appropriate). The results were analysed according to the RAND/UCLA Appropriateness Method. RESULTS: Forty-five participants from 13 countries (on three continents) participated. After the first round, a consensus was obtained for 82.5% of the 103 initially proposed statements. After the second round, a final consensus was obtained for 102 statements. CONCLUSIONS: We have reached an international Delphi-based consensus on best supportive care practice for SJS/TEN. Our expert consensus should help guide physicians in treating patients with SJS/TEN and thereby improve short-term prognosis and the risk of sequelae.

Clinical diversity and treatment approaches to blastic plasmacytoid dendritic cell neoplasm: a retrospective multicentre study.

BACKGROUND: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive type of haematologic precursor malignancy primarily often manifesting in the skin. We sought to provide a thorough clinical characterization and report our experience on therapeutic approaches to BPDCN. METHODS: In the present multicentric retrospective study, we collected all BPDCN cases occurring between 05/1999 and 03/2018 in 10 secondary care centres of the German-Swiss-Austrian cutaneous lymphoma working group. RESULTS: A total of 37 BPDCN cases were identified and included. Almost 90% of the patients had systemic manifestations (bone marrow, lymph nodes, peripheral blood) in addition to skin involvement. The latter presented with various types of cutaneous lesions: nodular (in more than 2/3) and bruise-like (in 1/3) skin lesions, but also maculopapular exanthema (in circa 1/6). Therapeutically, 22 patients received diverse combinations of chemotherapeutic regimens and/or radiotherapy. Despite initial responses, all of them ultimately relapsed and died from progressive disease. Eleven patients underwent haematopoietic stem cell transplantation (HSCT; autologous HSCT n = 3, allo-HSCT n = 8). The mortality rate among HSCT patients was only 33.33% with a median survival time of 60.5 months. CONCLUSION: Our study demonstrates the clinical diversity of cutaneous BPDCN manifestations and the positive development observed after the introduction of HSCT.

Analysis of anti-tumour necrosis factor-induced skin lesions reveals strong T helper 1 activation with some distinct immunological characteristics.

BACKGROUND: Psoriasiform and eczematous eruptions are the most common dermatological adverse reactions linked to anti-tumour necrosis factor (TNF)-α therapy. Yet, a detailed characterization of their immune phenotype is lacking. OBJECTIVES: To characterize anti-TNF-α-induced inflammatory skin lesions at a histopathological, cellular and molecular level, compared with psoriasis, eczema (atopic dermatitis) and healthy control skin. METHODS: Histopathological evaluation, gene expression (quantitative real-time polymerase chain reaction) and computer-assisted immunohistological studies (TissueFAXS) were performed on 19 skin biopsies from patients with inflammatory bowel disease (n = 17) and rheumatoid arthritis (n = 2) with new-onset inflammatory skin lesions during anti-TNF-α-therapy. RESULTS: Although most biopsies showed a psoriasiform and/or spongiotic (eczematous) histopathological architecture, these lesions were inconsistent with either psoriasis or eczema on a molecular level using an established chemokine (C-C motif) ligand 27/inducible nitric oxide synthase classifier. Despite some differences in immune skewing depending on the specific histopathological reaction pattern, all anti-TNF-α-induced lesions showed strong interferon (IFN)-γ activation, at higher levels than in psoriasis or eczema. IFN-γ was most likely produced by CD3/CD4/Tbet-positive T helper 1 lymphocytes. CONCLUSIONS: New-onset anti-TNF-α-induced eruptions previously classified as psoriasis or spongiotic dermatitis (eczema) exhibit a molecular profile that is different from either of these disorders.

[Skin barrier defects in atopic dermatitis: new treatments?]. / Anomalies de la barrière cutanée dans la dermatite atopique: une piste pour de nouveaux traitements?

Atopic dermatitis (AD) is a chronic inflammatory skin disorder and the most frequent skin disease in children. Skin barrier defects play a crucial role in its pathogenesis. 50% of patients suffering from AD present mutations in the filaggrin gene, coding for a key protein of the upper layer of the skin. However these mutations alone are not sufficient for disease development, suggesting that environmental factors are also of great importance in the genesis of AD. In particular skin infections frequently provoke clinical exacerbations in patients suffering from AD. New insights into skin barrier dysfunctions have facilitated the development of drugs targeting the sustainable restitution of the skin's physiologic function. These agents could modify the pharmacological approach of AD treatments in the future.

Plasma levels of nucleosomes and nucleosome-autoantibody complexes in murine lupus: effects of disease progression and lipopolyssacharide administration.

OBJECTIVE: To evaluate the effect of disease progression and lipopolysaccharide (LPS) administration on the presence of nucleosomes, antinucleosome reactivity, and nucleosome-Ig complexes in the circulation of MRL and control mice. METHODS: Plasma samples from lupus-prone (MRL/lpr and MRL/+) and control (CBA, Swiss, and BALB/c) mice were tested in enzyme-linked immunosorbent assays for the presence of nucleosomes, antinucleosome antibodies, and nucleosome-Ig complexes. Nucleosome kinetics, apoptosis induction, and phagocytosis of apoptotic cells were also analyzed in MRL/lpr, MRL/+, and CBA control mice after a single injection of LPS or phosphate buffered saline. RESULTS: Nucleosomes were found in the circulation of MRL/lpr and MRL/+ mice from week 4 onward. Nucleosomes were also detected in young control mice, but with increasing age, the nucleosomes disappeared. Antinucleosome antibodies, nucleosome-Ig complexes, and albuminuria were found only in the MRL/lpr mice. LPS administration led to a significant increase in circulating nucleosomes (3-8-fold) in all strains tested. In only the MRL/lpr mice was this increase followed by a significant decrease in antinucleosome titers and an increase in nucleosome-Ig complexes. The number of apoptotic cells in the thymus after LPS was significantly higher in the MRL/lpr mice than in the MRL/+ and CBA control mice. LPS caused a profound reduction (50-70%) of the phagocytosis of apoptotic cells by peritoneal macrophages, which was comparable for all strains. CONCLUSION: In MRL lupus-prone mice, nucleosomes are persistently present in the circulation, whereas in control mice, nucleosomes are present only at a young age. The formation of antinucleosome antibodies and nucleosome-Ig complexes is a characteristic feature of MRL/lpr mice. LPS administration increases systemic nucleosome release due to an enhancement of apoptosis and a decrease in the clearance of apoptotic cells.

Histone-containing immune complexes are to a large extent responsible for anti-dsDNA reactivity in the Farr assay of active SLE patients.

Increased titres of anti-dsDNA antibodies, especially if of high avidity, are associated with renal exacerbations in patients with systemic lupus erythematosus (SLE). One of the most reliable assays to measure anti-dsDNA antibodies, the Farr assay, is believed to detect preferentially high avidity antibodies. Purified non-complexed monoclonal antibodies (mAbs) against nucleosomes, obtained from mice with SLE, are not reactive in the Farr assay, but can become so once complexed to nucleosomes. These Farr-positive, nucleosome containing, immune complexes were also able to bind in vivo to the glomerular basement membrane (GBM), predominantly via heparan sulphate (HS). To evaluate whether in SLE patients the same kind of immune complexes are responsible for Farr reactivity, IgG from serum or plasma was isolated under dissociating and physiological conditions. We observed that after purification under dissociating conditions, Farr reactivity was significantly decreased (P<0.0001) in contrast to reactivity with histones and two 'control' antigens: Epstein Barr Virus (EBV) and Ro/SS-A. Reactivity with nucleosomes also decreased after purification, although to a lesser extent. Plasma purified under physiological conditions showed no decrease in Farr reactivity. The importance of histones for the generation of immune complexes is supported by the two following observations. Firstly, the presence of histones could be demonstrated in serum and plasma of SLE patients but not in serum of healthy controls or in IgG preparations purified under dissociating conditions. Secondly, Farr reactivity of purified IgG preparations could be restored by addition of purified histones. From these studies we conclude that histones containing immune complexes are responsible for a large part of the Farr reactivity in active SLE, and are therefore indirectly implicated in the pathogenesis of lupus nephritis.

Role of nucleosomes for induction and glomerular binding of autoantibodies in lupus nephritis.

The cardinal feature of systemic lupus erythematosus is the formation of anti-nuclear antibodies. In recent years, it has become clear that the nucleosome is a major autoantigen that drives this T cell-dependent autoimmune response, as exemplified by the presence of nucleosome-specific T helper cells and the high prevalence of nucleosome-specific autoantibodies. The only way to generate nucleosomes in vivo is by the process of apoptosis. There is growing evidence that in systemic lupus erythematosus apoptosis is disturbed, leading to the release of nucleosomes. Moreover, apoptosis-induced modifications of these autoantigens may render them more immunogenic, especially if the removal of apoptotic cells is insufficient. The first indications for the impaired clearance of apoptotic cells in systemic lupus erythematosus are emerging. Nucleosomes are also important for mediating tissue lesions, especially glomerulonephritis. In lupus nephritis nucleosomes, nucleosome-specific antibodies and nucleosome/IgG complexes have been identified in the glomerular immune deposits. Via their cationic histone part nucleosomes mediate the binding of anti-nuclear antibodies to intrinsic constituents of the glomerular basement membrane, such as the anionic heparan sulfate and collagen IV. Appreciation of this binding mechanism may lead to new treatment strategies, as shown for non-coagulant heparinoids.

Decreased staining of heparan sulfate in non-lesional skin of a subgroup of patients with systemic lupus erythematosus.

Heparan sulfate proteoglycans (HSPGs) are components of the basement membrane of various tissues. They are composed of a core protein and of the negatively charged glycosaminoglycan side chain heparan sulfate, which is covalently bound to the core protein. We previously found that in both human and murine lupus nephritis, heparan sulfate staining in the basement membrane of the glomerulus is almost completely absent, and that there was an inverse correlation between heparan sulfate staining and glomerular immunoglobulin deposits. As immunoglobulin deposits are also present in the skin of systemic lupus erythematosus patients, we investigated the heparan sulfate staining pattern in the basement membrane of the dermal-epidermal junction. furthermore, we studied whether there was a correlation between heparan sulfate staining and deposition of immunoglobulin in the basement membrane of this junction, and between heparan sulfate staining and the presence of anti-DNA antibodies in the serum. Biopsies of non-lesional skin of 21 systemic lupus erythematosus patients (15 anti-DNA positive and 6 anti-DNA negative patients at the time of biopsy) were stained for the HSPG-core protein (mAb JM-72), highly sulfated stretches within heparan sulfate (JM-13), the low sulfated regions of heparan sulfate (mAb JM-403) and for immunoglobulin depositions. Abnormal and discontinuous staining of the low sulfated parts of heparan sulfate using mAb JM-403 in the basement membrane was found in skin biopsies of 4 out of 15 systemic lupus erythematosus patients with anti-DNA antibodies. In contrast, all specimens of anti-DNA negative patients showed normal continuous staining. Staining with JM-13 and JM-72 showed normal linear staining in both groups. The decreased heparan sulfate staining was correlated significantly with the presence of immunoglobulin deposits in the basement membrane. The subgroup could not be identified by its clinical picture. Our results suggest similar but not identical pathways in systemic lupus erythematosus nephritis and skin of systemic lupus erythematosus patients.

Attenuation of murine lupus nephritis by mycophenolate mofetil.

Mycophenolate mofetil (MMF) is the morpholinoethyl ester of mycophenolic acid, which is its active metabolite. MMF is effective in prolonging survival of allografts and xenografts. However, little is known about the effects and the main mechanism of action of MMF in autoimmune diseases. In this study, the effect of MMF on the spontaneous disease progression in the MRL/lpr mouse model of lupus was examined. Eight-week-old MRL/lpr mice (n=18) were orally treated with MMF dissolved in a vehicle (90 mg/kg) once a day. Control animals received vehicle alone (n=17). The incidence of albuminuria (>300 microg/18 h) was significantly reduced by MMF treatment compared with vehicle-treated controls (cumulative incidence of albuminuria at 23 wk in MMF-treated mice; 22% versus 88% in controls; P=0.0001). The glomerulonephritis was histologically less severe in MMF-treated mice than in control mice (P=0.005). Furthermore, in immunofluorescence studies the amount of immunoglobulin and C3 deposits in the glomerular capillary wall was significantly less in MMF-treated mice (P < or = 0.002). Surprisingly, in vivo no clear-cut immune-modulating effects were observed because there were no differences between MMF-treated and control animals with regard to autoantibody formation. Also, spleen enlargement and numbers of CD3+, CD4+, and CD8+ T cells in spleen, lymph nodes, and peripheral blood were not different between both groups. Furthermore, no immunosuppressive properties of 90 mg/kg MMF were found in BALB/c mice on delayed-type hypersensitivity and primary antibody response to methylated bovine serum albumin. Interestingly, renal perfusion experiments revealed that binding of nucleosome/antinucleosome complexes to the glomerular basement membrane is decreased in MMF-treated mice compared with control mice. It is concluded that MMF suppresses the development of lupus glomerulonephritis and albuminuria in MRL/ lpr mice. The observed reduction of glomerular immunoglobulin deposits in MMF-treated mice and the renal perfusion studies indicate that MMF treatment leads to a decreased binding of immune complexes in the glomerular capillary wall in lupus nephritis.

Antigen specificity of anti-nuclear antibodies complexed to nucleosomes determines glomerular basement membrane binding in vivo.

Monoclonal anti-nuclear antibodies which are complexed to nucleosomes are able to bind to the glomerular basement membrane (GBM) in vivo, whereas purified antibodies do not bind. The positively charged histone moieties in the nucleosome are-responsible for the binding to anionic determinants in the GBM. We tested the hypothesis that the specificity of the autoantibodies complexed to the nucleosome influences the glomerular binding of the antibody-nucleosome complex. We induced the formation of these immune complexes in vivo, by intraperitoneal inoculation of hybridomas producing monoclonal anti-nuclear antibodies (four anti-histone, three anti-double stranded (ds)DNA and three anti-nucleosome antibodies) into nude BALB/c mice. In ascites and plasma from the mice inoculated with these hybridomas, nucleosome/autoantibody complexes were detected in comparable amounts. Immunofluorescence of kidney sections revealed that about 60% of the mice inoculated with anti-nucleosome or anti-dsDNA hybridomas had immunoglobulin deposits in the GBM, whereas only 15% of the mice with anti-histone hybridomas showed these deposits (p < or = 0.04). In the Matrigel-ELISA (used as a GBM surrogate) ascites from anti-nucleosome or anti-DNA hybridomas displayed significantly higher titers (p < or = 0.002) than ascites from anti-histone hybridomas. In conclusion, nucleosome/immunoglobulin complexes comprising anti-nucleosome or anti-dsDNA auto-antibodies do bind more frequently to the GBM in vivo than nucleosome/immunoglobulin complexes containing anti-histone antibodies. It therefore appears that the specificity of the antibody bound to the nucleosome is a critical determinant for the nephritogenic potential of the nucleosome-autoantibody complex.

Nucleosomes and histones are present in glomerular deposits in human lupus nephritis.

BACKGROUND: Recently we showed that antinuclear autoantibodies complexed to nucleosomes can bind to heparan sulphate (HS) in the glomerular basement membrane (GBM) via the histone part of the nucleosome. Histones have been identified in glomerular deposits in human and murine lupus nephritis. In addition, a decreased HS staining in the GBM was found, most probably due to masking by deposition of antibodies complexed to nucleosomes. METHODS: In this study we first investigated whether histones or nucleosomes could be identified in glomerular deposits in human lupus nephritis, and secondly whether the presence of these nuclear components was correlated with absence of HS staining. Kidney biopsies of SLE patients (11 with diffuse proliferative glomerulonephritis (DPGN) and six with membranous glomerulonephritis (MGN)) and non-SLE glomerular diseases were stained for histones. DNA, nucleosomes, IgG and HS. RESULTS: Using a polyclonal anti-H3 1 21 antiserum, histones were detected in all patients with DPGN and in two of six patients with SLE-MGN (P < 0.01). Using a monoclonal antihistone antibody, histones were stained in three patients with DPGN, but in none of the biopsies with MGN. Using nucleosome specific monoclonal antibodies, nucleosomes were detected in five patients with DPGN, in two patients with MGN, but in none of the biopsies with non-SLE glomerulonephritis. HS staining was nearly absent in DPGN, whereas staining was only moderately reduced in patients with MGN and controls (P = 0.001). CONCLUSION: Using polyclonal and monoclonal antihistone antisera, histones were identified in all patients with DPGN and their presence was associated with a decrease of HS staining. Nucleosomes were identified in five of 11 patients with DPGN and in two of six patients with MGN. This is the first demonstration of nucleosomes in glomerular deposits in SLE nephritis.

Specificity of monoclonal anti-nucleosome auto-antibodies derived from lupus mice.

Recently, anti-nucleosome antibodies, which do not bind to DNA or to individual histones, have been identified in longitudinal studies in lupus mice. These anti-nucleosome antibodies occur early in spontaneous SLE and are formed prior to other anti-nuclear specificities. However, nucleosomal epitopes are yet to be fully characterized. We selected a panel of six monoclonal anti-nucleosome antibodies (mAbs) (#2, #32, #34, PL2-6, LG8-1 and LG10-1) derived from lupus mice. These mAbs were tested in ELISA on subnucleosome structures and on a panel of 53 histone peptides, covering the entire sequence of the five histones. Two mAbs reacted with one of these peptides, but the reactivity hardly exceeded the background reactivity. Based on the nucleosome and subnucleosome ELISA we identified different recognition patterns. Three mAbs showed the highest reactivity towards the intact nucleosome. For two of them (#32 and LG8-1) the nucleosomal epitope was primarily located on H2A-H2B/DNA, whereas for mAb #34 this primary epitope was located on H3/H4/DNA. Two mAbs (#2 and PL2-6) showed the highest reactivity with H2A-H2B/DNA and one mAb (LG10-1) recognized H3-H4/DNA. In the subnucleosome ELISA all but one (mAb #32) recognized more than one epitope, including DNA complexed to a variety of cationic molecules. Comparing these reactivities we identified for all mAbs one specific nucleosomal epitope, whereas reactivity with other subnucleosomes was comparable to the reactivity towards DNA complexed with cationic molecules. In inhibition experiments both in ELISA and in immunofluorescence it was found that only one of the mAbs (i.e. PL2-6), recognizing an epitope on H2A-H2B/DNA as primary epitope, could be inhibited by H2A-H2B/DNA in fluid phase. The two mAbs recognizing an epitope on H3-H4/DNA as primary epitope could be inhibited by H3-H4/DNA in fluid phase. From these analyses, we conclude first that for these nucleosome specific mAbs linear histone peptides are not very important. Second, that these mAbs all recognize different epitopes on both H2A/H2B-DNA and H3/H4-DNA and third that some solid phase H2A/H2B-DNA epitopes are not expressed on fluid phase H2A/H2B-DNA. Our findings suggest that in SLE the nucleosome can act as auto-antigen and that there is no immunodominant beta cell epitope within the nucleosome.

Heparin and heparinoids prevent the binding of immune complexes containing nucleosomal antigens to the GBM and delay nephritis in MRL/lpr mice.

Monoclonal anti-nucleosome antibodies (mAbs) complexed to nucleosomal antigens can bind to DNA and to heparan sulfate (HS) in ELISA and to the GBM in vivo in a rat renal perfusion system, whereas non-complexed mAbs do not bind [1]. In this study, we analyzed whether heparin (HEP) or N-desulfated/acetylated heparins (DSA-HEP), structurally and functionally strongly related to HS, are able to prevent the binding of these complexed mAbs to DNA and to HS in vitro and to rat GBM in vivo. In ELISA the binding of nucleosome complexed antinucleosome antibodies to DNA and HS was inhibited dose-dependently by HEP, DSA-HEP and low molecular weight (LMW) DSA-HEP. Intravenous injection of nucleosome/anti-nucleosome immune complexes without heparin/heparinoids in BALB/c mice led to GBM binding, while simultaneous injection of heparin/heparinoids with complexed antibodies or pretreatment with heparin subcutaneously prior to injection of complexes prevented this binding. Subsequently, we tested the preventive effect of HEP, DSA-HEP and LMW-DSA-HEP on progression of renal disease in MRL/lpr mice. Treatment was started at an age of eight weeks in a dose of 50 micrograms daily. With all three drugs albuminuria was significantly delayed compared to PBS treated controls (cumulative incidence of proteinuria at 20 weeks in controls 60% vs. 13%, 14% and 6% respectively for HEP, DSA-HEP and LMW-DSA-HEP; P < 0.05). At week 21 the glomerulonephritis was histologically less severe in heparin/heparinoid treated animals (P = 0.02). In immunofluorescence the amount of immunoglobulin and C3 deposits in the glomerular capillary wall tended to be less in heparin/heparinoid treated mice compared to PBS treated controls (P = 0.07). Furthermore, at 20 weeks anti-HS levels in plasma of heparin/heparinoid treated mice were significantly lower (P < 0.05). We conclude that interaction of heparin or heparin analogs with HS reactive immune complexes containing nucleosomal antigens prevents the binding of these immune complexes to the GBM and delays nephritis in MRL/lpr mice.

Significance of anti-nuclear and anti-extracellular matrix autoantibodies for albuminuria in murine lupus nephritis; a longitudinal study on plasma and glomerular eluates in MRL/l mice.

The relationship between autoantibody reactivities and nephritis in systemic lupus erythematosus (SLE) is unclear. We studied MRL/l mice which developed a considerable albuminuria (either mice with short ( < 1 week) or heavy and prolonged (3 weeks) albuminuria) and compared them with non-albuminuric age-matched controls, with young (12 weeks old) non-albuminuric mice and with mice which were followed for 36 weeks and did not develop albuminuria. In a longitudinal prospective study on plasma samples we correlated a variety of anti-nuclear reactivities and reactivities against extracellular matrix (ECM) components, with the onset of albuminuria. We found that at the onset of albuminuria, anti-DNA was higher while anti-nucleosome and anti-H2A/H2B-DNA subnucleosome reactivities were lower compared with age-matched non-albuminuric mice. We also studied glomerular eluates of these mice in ELISA and in indirect immunofluorescence (IF). In the eluates we found with IF that anti-glomerular basement membrane (GBM)-tubular basement membrane (TBM) antibodies were already present in 12-week-old non-albuminuric mice. These eluates showed no anti-nuclear antibodies. In eluates of albuminuric mice more immunoglobulin was deposited, and anti-ECM, anti-DNA and anti-nucleosome reactivities were higher than in eluates of age-matched non-albuminuric mice. The deposition of anti-nucleosome antibodies preceded the deposition of anti-DNA antibodies since they were deposited to a greater extent in mice with a short albuminuria. We conclude that anti-GBM-TBM antibodies are the first autoantibodies that deposit in glomeruli of MRL/l mice at an early age. The onset of albuminuria is associated with additional deposition of both anti-ECM and anti-nuclear (anti-nucleosome and anti-DNA) antibodies, but the difference with non-albuminuric mice seems to be more quantitative than qualitative.

In vivo ANA is a fixation artifact: nucleosome-complexed antinucleosome autoantibodies bind to the cell surface and are internalized.

It has been suggested that binding of anti-double-standed DNA antibodies to cell surfaces, followed by internalization and nuclear binding (so called in vivo ANA) is of pathophysiological significance for tissue damage in systemic lupus erythematosus. We have shown before that pathogenic antinuclear antibodies complexed to nucleosomal antigens can bind to heparan sulfate in the glomerular basement membrane in vivo. Because nucleosomes are also reported to bind to the cell surface, we hypothesized that in vivo ANA is a property of antinuclear antibodies bound to nucleosomal antigens. Therefore, we studied three antinucleosome monoclonal antibodies (mAb) that exhibit in vivo ANA as seen by immunofluorescence in mice inoculated intraperitoneally with the hybridoma producing the mAb. The same mAb complexed to nucleosomal antigens after intravenous injection into mice induced in vivo ANA, in contrast to purified noncomplexed mAb. To study this in more detail, we incubated complexed mAb with various cell lines and found binding to the cell surface and subsequent internalization into cytoplasmic vesicles. However, no binding to the nucleus was observed by immunoelectron microscopy (IEM) and confocal laser microscopy. Noncomplexed mAb did not bind to the cell surface. Next, from mice bearing the hybridomas producing the mAb intraperitoneally, a small part of the kidney was snap frozen in liquid N2, fixed with acetone, and studied in immunofluorescence, whereas the remaining part of the kidney was fixed in vivo by renal perfusion with a mixture of 0.01 M sodium periodate, 0.075 M lysine HCl, 0.0375 M Na2HPO4, and 2% paraformaldehyde (PLP) and studied in both immunofluorescence and IEM. In the acetone-fixed kidney sections obtained without in vivo fixation we again observed in vivo ANA. However, after in vivo PLP perfusion fixation, no nuclear binding was found. In IEM, localization in cytoplasmic vesicles was seen. In conclusion, antinucleosome antibodies complexed to nucleosomal antigens can bind to the cell surface and are transported into the cytoplasm, but do not bind to the nucleus. The reported nuclear localization of antinuclear antibodies is caused by a fixation artifact.