The embryotoxicity of sera from patients with autoimmune diseases on post-implantation rat embryos in culture persists during remission and is not related to oxidative stress.

We evaluated the embryolethality and embryotoxicity of sera from patients suffering from autoimmune diseases during remission on post-implantation rat embryos cultured on these sera and determined the association between the patients' clinical history, high blood levels of specific antibodies, medications, and oxidative stress parameters. One hundred and eighty, 10.5-day-old rat embryos were cultured in their yolk sacs in 33 sera of systemic lupus erythematosus (SLE)/antiphospholipid syndrome (APS) patients, and compared with 84 embryos cultured in rat sera and 88 embryos cultured in control human sera. The sera proved to be lethal and embryotoxic but not teratogenic resulting in smaller yolk sacs and embryos, lower protein level and lower developmental score. Significantly less embryos cultured in 'toxic' SLE/APS sera had peak 2 of low molecular weight antioxidants (LMWA) wave, implying a delayed maturation of the antioxidant defense. Lower peak 1 of LMWA correlated with a history of recurrent abortions. Embryonic levels of superoxide dismutase (SOD) and catalase (CAT) did not correlate with sera toxicity, patients' clinical history or specific antibodies. We conclude that SLE/APS patients' clinical remission did not prevent death or developmental delay accompanied by later appearance of peak 2 of LMWA in post-implantation rat embryo cultures. The normal levels of the antioxidant enzymes evaluated may indicate that sera toxicity is not related to oxidative stress.

Early diagnosis and treatment of atrioventricular block in the fetus exposed to maternal anti-SSA/Ro-SSB/La antibodies: a prospective, observational, fetal kinetocardiogram-based study.

BACKGROUND: A fetus exposed to maternal anti-SSA/Ro or anti-SSB/La antibodies (or both) may develop complete atrioventricular block (AVB), which results in high prenatal and postnatal morbidity and mortality. Until recently, only high-grade AVB could be diagnosed in utero. The tissue velocity-based fetal kinetocardiogram (FKCG) enables accurate measurement of AV conduction time and diagnosis of low-grade AVB. In the present multicenter observational study, we used FKCG to detect first-degree AVB in fetuses at risk. METHODS AND RESULTS: FKCG was performed in 70 fetuses of 56 mothers who were positive for anti-SSA/Ro and/or anti-SSB/La. Fetuses were monitored with weekly FKCG from 13 to 24 weeks' gestation, followed by monthly assessments until delivery in unaffected fetuses and weekly assessments in affected fetuses. AV conduction in 70 at-risk and 109 normal fetuses was compared. FKCG was obtained readily in all fetuses; 6 showed first-degree AVB (AV conduction time >2 z scores above normal mean) at 21 to 34 gestational weeks. Immediate maternal treatment with dexamethasone resulted in normalization of AV conduction in all affected fetuses within 3 to 14 days. AV conduction time in the remaining 64 untreated fetuses remained normal throughout gestation. The ECG PR interval immediately after birth was normal in all affected newborns. No child developed AVB or cardiomyopathy in the subsequent 1- to 6-year (median 4-year) follow-up. CONCLUSIONS: The present findings suggest that an FKCG can detect first-degree AVB in the fetus exposed to maternal anti-SSA/Ro or anti-SSB/La antibodies (or both). Dexamethasone given on detection was associated with normalized AV conduction in fetuses with first-degree AVB. No fetus in the present study developed complete prenatal or postnatal AVB.

'Danger' effect of low-density lipoprotein (LDL) and oxidized LDL on human immature dendritic cells.

Dendritic cell (DC) maturation may accelerate autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, and may contribute to accelerated atherosclerosis seen in these patients. The immune system responds to both exogenous and endogenous 'dangerous' signals that can induce dendritic cell maturation. We have found that autologous plasma contains danger signals that induce up-regulation of major histocompatibility complex (MHC) class II and co-stimulatory molecules in immature DCs (iDCs). The objective of this study was to determine whether low-density lipoprotein (LDL) and/or oxidized LDL (oxLDL) constitute danger signals, and to assess the effect of exposure to LDL and oxLDL following monocyte differentiation into iDCs in lipoprotein-deficient serum (LPDS). IDCs were generated in the presence of autologous plasma or LPDS. Expression of maturation and migration molecules was evaluated using flow cytometry, and morphology was assessed by light microscopy. Pro- or anti-apoptotic effect was determined using annexin V and propidium iodide binding. Phagocytosis of apoptotic cells was evaluated using autologous plasma or LPDS. LDL and oxLDL were clearly able to slightly up-regulate levels of HLA-DR and co-stimulatory molecule CD86. High oxLDL concentrations (50-100 microg/ml) were associated with expression of additional maturation molecules. Moreover, iDCs that were prepared in LPDS showed partial maturation following exposure to LDL and oxLDL, and improved tolerogenic apoptotic cell uptake. This study suggests that oxLDL, and to some extent LDL, are at least partly responsible for the iDC 'danger' response induced by autologous plasma.

Reduced placental growth and hCG secretion in vitro induced by antiphospholipid antibodies but not by anti-Ro or anti-La: studies on sera from women with SLE/PAPS.

Systemic lupus erythematosus (SLE) and primary anti-phospholipid syndrome (PAPS) are autoimmune diseases causing recurrent pregnancy loss. We hypothesized that anti-phospholipid antibodies (aPL), but not anti-Ro and anti-La antibodies, might have a role through direct placental damage. We cultured human placental explants in sera from women with SLE/PAPS with different antibodies. These sera were found to reduce placental growth and increase trophoblastic apoptosis. No effect was found on estradiol or progesterone secretion, but inhibition in betahCG secretion was detected. BetahCG was reduced in women with a history of recurrent pregnancy loss or thromboembolic events, and was also the most sensitive marker when examining the effects of specific antibodies. High titers of aPL were found to cause the largest reduction in betahCG. Anti-Ro and anti-La did not induce placental damage. A strong correlation was found between the rise in the number of different antibodies in the sera and the incidence of recurrent pregnancy loss, which was also accompanied by a decline in the betahCG levels. In conclusion, aPL, but not anti-Ro or anti-La, may cause placental damage in vitro. Thus betahCG levels might constitute a predictive marker for the risk of placental damage and pregnancy loss in women with SLE/PAPS.

Outcome of a national Israeli cohort of pediatric systemic lupus erythematosus.

The aim of this study was to describe the clinical manifestations and outcomes of a national cohort of childhood systemic lupus erythematosus (cSLE). All cases of cSLE registered in the Israeli national registry of children with rheumatic diseases between 1987-2003 were examined for disease activity and damage by the SLE disease activity index (SLEDAI) and SLE collaborating clinics/American College of Rheumatology (SLICC/ACR) damage index. Demographic, clinical, laboratory and treatment factors were analysed for their effect on the outcome. One-hundred and two patients were identified, 81% females, with a mean age at diagnosis of 13.3 +/- 2.6 years. The mean SLEDAI score was 17.2 +/- 9.0 (range 2-60). Fifty four patients were followed for at least five years. The mean SLEDAI decreased to 7.6 +/- 6.3 (0-29) and the mean SLICC/ACR damage index was 0.7 +/- 1.6 (0-8). Five patients developed chronic renal failure. No patients died. No factors were found to be significantly associated with the outcome except the initial SLEDAI score. The five-year outcome of our national cSLE cohort was good; with relatively low activity and minimal damage in most patients. The initial SLEDAI predicted the development of late damage.

The UVB-induced gene expression profile of human epidermis in vivo is different from that of cultured keratinocytes.

In order to obtain a comprehensive picture of the molecular events regulating cutaneous photodamage of intact human epidermis, suction blister roofs obtained after a single dose of in vivo ultraviolet (UV)B exposure were used for microarray profiling. We found a changed expression of 619 genes. Half of the UVB-regulated genes had returned to pre-exposure baseline levels at 72 h, underscoring the transient character of the molecular cutaneous UVB response. Of special interest was our finding that several of the central p53 target genes remained unaffected following UVB exposure in spite of p53 protein accumulation. We next compared the in vivo expression profiles of epidermal sheets to that of cultured human epidermal keratinocytes exposed to UVB in vitro. We found 1931 genes that differed in their expression profiles between the two groups. The expression profile in intact epidemis was geared mainly towards DNA repair, whereas cultured keratinocytes responded predominantly by activating genes associated with cell-cycle arrest and apoptosis. These differences in expression profiles might reflect differences between mature differentiating keratinocytes in the suprabasal epidermal layers versus exponentially proliferating keratinocytes in cell culture. Our findings show that extreme care should be taken when extrapolating from findings based on keratinocyte cultures to changes in intact epidermis.

Quinidine and procainamide inhibit murine macrophage uptake of apoptotic and necrotic cells: a novel contributing mechanism of drug-induced-lupus.

UNLABELLED: A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined. OBJECTIVE: To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL. METHODS: Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis. RESULTS: Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen. CONCLUSION: Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL.

Methotrexate related adverse effects in patients with rheumatoid arthritis are associated with the A1298C polymorphism of the MTHFR gene.

BACKGROUND: There is an association between C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene and methotrexate related toxicity. OBJECTIVE: To examine the relations between the recently described A1298C polymorphism of the MTHFR gene, plasma homocysteine, methotrexate toxicity, and disease activity in patients with rheumatoid arthritis. DESIGN: A cross sectional study on 93 methotrexate treated patients with rheumatoid arthritis, comprising a clinical interview and physical examination to determine disease activity and methotrexate related adverse reactions. Genotype analysis of the MTHFR gene was carried out and fasting plasma homocysteine and serum folate concentrations were measured. The data were analysed using univariate analysis. Allele and genotype distributions were compared with those of a healthy control group. RESULTS: The frequency of the 1298CC genotype (24.7%) in the rheumatoid study group was greater than expected in the general population (12.8%, p<0.001). This genotype was associated with a significantly low rate of methotrexate related side effects. The odds ratio for side effects in patients with wild type 1298AA genotype v 1298CC genotype was 5.24 (95% confidence interval, 1.38 to 20). No correlation of disease activity variables or plasma homocysteine with MTHFR A1298C and C677T polymorphisms was observed. CONCLUSIONS: 1298CC polymorphism was more common in methotrexate treated rheumatoid patients than expected in the population, and was associated with a reduction in methotrexate related adverse effects. The A1298C polymorphism of the MTHFR gene may indicate a need to adjust the dose of methotrexate given to patients with rheumatoid arthritis.

Accelerated Fas-mediated apoptosis of monocytes and maturing macrophages from patients with systemic lupus erythematosus: relevance to in vitro impairment of interaction with iC3b-opsonized apoptotic cells.

Impaired handling of apoptotic cells has been suggested as an important factor in the development of systemic lupus erythematosus (SLE), and a role for complement in the removal of apoptotic cells was shown recently. We studied the in vitro function of macrophages from 40 patients with SLE and their matched controls in the removal of heterologous apoptotic cells opsonized by iC3b. Interaction index of apoptotic cells opsonized by iC3b was significantly lower in patients with SLE and averaged 71% +/- 37 of that of healthy individuals (p < 0.002) and 69% +/- 35 of patients with rheumatoid arthritis (p < 0.007). SLE patients had increased apoptosis of both freshly isolated monocytes (p < 0.001) and maturing macrophages (p < 0.04) that led to decreased density of monocyte-derived macrophages. Apoptosis was inhibited by adding soluble Fas receptor indicating Fas-mediated apoptosis. As demonstrated in both healthy controls and patients with SLE, decreased macrophage density by itself caused significant decreased uptake of apoptotic cells by the remaining macrophages. Maintaining normal density in SLE patients either by an increased initial density or by using soluble Fas restored the interaction capacity of the individual macrophages in the majority of patients. We concluded that impaired in vitro interaction of iC3b-opsonized apoptotic cells with macrophages from patients with SLE was mainly associated with Fas-dependent accelerated apoptosis of the monocytes/macrophages. Accelerated apoptosis of phagocytes may represent a novel in vitro mechanism of impairment of interaction with apoptotic cells that, apart from reducing the number of professional phagocytes, alters the function of the remaining macrophages.

Complement and apoptosis.

Apoptosis and necrosis are two forms of cell death characterized by distinct morphologies. Until recently, complement-mediated cell lysis has been presented as a classical example of necrotic cell death. However, recent reports on apoptogenic effects of complement have shaken this dogma. The field has become even more confusing with descriptions of anti-apoptotic effects of complement. Necrosis has been associated traditionally with inflammation, whereas apoptosis has been regarded as noninflammatory. Therefore, first descriptions of the capacity of the complement system to identify apoptotic cells and to be activated by them, led to the development of the concept that complement opsonizes apoptotic cells for fast clearance by phagocytic cells. In the absence of such opsonization, (eg in C1q or C4 deficiency), apoptotic cells may remain longer in the body and may stimulate autoantibody production or undergo pro-inflammatory secondary necrosis. This has been associated in man and mouse with the development of an autoimmune disease like systemic lupus erythematosus. However, complement may also interfere with the programmed intention of apoptosis to avoid triggering of inflammation. Our recent results show that, under specific conditions, early apoptotic cells are not only opsonized by complement but may also be lysed, raising the possibility that under certain conditions apoptosis will be associated with an inflammatory reaction. The review describes and discusses the reports covering the various aspects of the interface between complement and apoptosis and its possible relevance to autoimmune diseases and inflammation and raises the following questions: 1. Can activated complement proteins induce apoptotic cell death? 2. Can complement protect cells from apoptosis? 3. Do apoptotic cells activate complement? and 4. What role is complement playing in clearance of apoptotic cells and which complement receptors are involved? Clearly, this research field is highly complex and still at its formation stage, yet interesting and important mechanisms are bound to emerge from these studies.

Opsonization of apoptotic cells. Implications for uptake and autoimmunity.

As a part of innate immunity, soluble host proteins called opsonins, which include complement ligands and immunoglobulins, initially coat microorganisms that penetrate the mammalian sterile milieu. The main purpose of opsonization is to allow subsequent clearance of opsonized particles by specific receptors on the surface of leukocytes. Similarly, several proteins that may act as opsonins and have a role in uptake of apoptotic cells and bodies include thrombospondin I, the complement system, beta 2GPI, immunoglobulins, CRP, and some unidentified others. The surface changes that lead to opsonization include the appearance of phosphatidylserine that acts as an activator molecule for some known opsonins as the complement system and beta 2GPI. The consequence of altered opsonization is demonstrated by the development of autoimmunity in C1q deficient mice, and the pro-inflammatory response by macrophages ingesting apoptotic cell opsonized by an autoantibody.

The immune response to apoptotic cells.

Programmed cell death (PCD) can be divided into two distinct but linked sequential processes, killing of the cells and removal of the dead cells, which may be a neighboring cell or a professional phagocyte. Following internalization of the apoptotic cell, the phagocyte typically triggers neither the development of a pro-inflammatory response nor the production of autoantibodies directed against apoptotic self antigens. Since apoptotic cells are characterized by translocation of autoantigens such as nucleosomes to the surface of the cell, we tested the hypothesis that excess or abnormally processed apoptotic cells can generate autoantibodies. We have found that syngeneic apoptotic load can induce transient hypergammaglobulinemia, anti-DNA, anticardiolipin, and glomerular depositions in normal mice. Furthermore, we also found that one of the important mechanisms of uptake of apoptotic cells involves opsonization by the complement system, suggesting that deficient states could lead to aberrant handling of apoptotic cells. Therefore, conditions in which apoptotic cells become immunogenic may explain antigen selection in inflammatory and autoimmune conditions, such as in systemic lupus erythematosus (SLE).

Complement-dependent clearance of apoptotic cells by human macrophages.

Apoptotic cells are rapidly engulfed by phagocytes, but the receptors and ligands responsible for this phenomenon are incompletely characterized. Previously described receptors on blood- derived macrophages have been characterized in the absence of serum and show a relatively low uptake of apoptotic cells. Addition of serum to the phagocytosis assays increased the uptake of apoptotic cells by more than threefold. The serum factors responsible for enhanced uptake were identified as complement components that required activation of both the classical pathway and alternative pathway amplification loop. Exposure of phosphatidylserine on the apoptotic cell surface was partially responsible for complement activation and resulted in coating the apoptotic cell surface with C3bi. In the presence of serum, the macrophage receptors for C3bi, CR3 (CD11b/CD18) and CR4 (CD11c/CD18), were significantly more efficient in the uptake of apoptotic cells compared with previously described receptors implicated in clearance. Complement activation is likely to be required for efficient uptake of apoptotic cells within the systemic circulation, and early component deficiencies could predispose to systemic autoimmunity by enhanced exposure to and/or aberrant deposition of apoptotic cells.

Systemic exposure to irradiated apoptotic cells induces autoantibody production.

During apoptotic cell death, cell surface ligands initiate phagocytosis of the dying cell. Clearance of these apoptotic cells is thought to occur without an immune response. Since a number of autoantigens are located at the cell surface or within apoptotic blebs, we examined whether exposure of mice to syngeneic apoptotic cells by the intravenous route could induce autoantibody production. Normal mice injected with syngeneic apoptotic thymocytes developed antinuclear autoantibodies and anticardiolipin and anti-ssDNA antibodies. The autoantibody levels were generally lower than those observed in MRL/Faslpr mice and were transient. Surprisingly, six out of six immunized mice demonstrated immunoglobulin G deposition in the glomeruli several months after immunization. These findings indicate that systemic exposure to apoptotic cells can induce an immune response in normal mice, and may help to explain antigen selection and initiation of the immune response in diseases characterized by increased rates of apoptosis such as AIDS and, possibly, systemic lupus erythematosus.

Anti-P antibodies and neuropsychiatric lupus erythematosus.

A high proportion of patients with SLE develop neuropsychiatric lupus during the course of their disease. The expression of disease varies significantly in terms of clinical manifestations, onset, and severity, so that this form of lupus remains a major diagnostic and therapeutic challenge. Because affected tissue cannot be sampled and animal models are not readily available, scientific investigation is considerably hampered. Approximately 15% of lupus patients have anti-P antibodies. These autoantibodies are highly specific for SLE. Most series show a highly significant association between anti-P and lupus psychosis, and this result is now confirmed in a series of 336 SLE patients. The mechanism explaining this association is uncertain and may simply reflect an immune response to damaged tissue. However, the possibility that the antibodies are pathogenic by directly binding to cell-surface receptors on neuronal cells or that they penetrate cells and inhibit protein synthesis within the cell requires further investigation. Whether T cells are involved in the pathogenesis of CNS disease is an important question that has received little attention. As in the CNS of patients with multiple sclerosis and patients with other autoimmune diseases, excess production of cytokines may contribute significantly to organ inflammation. We report associations between anti-P autoantibodies and certain MHC class II alleles, particularly HLA-DQB1*0602. These findings support a role for T cells in anti-P autoantibody production and encourage further studies of the role of autoantigen-specific T and B cells in injury to the central nervous system.