Activated protein C attenuates systemic lupus erythematosus and lupus nephritis in MRL-Fas(lpr) mice

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease leading to inflammatory tissue damage in multiple organs (e.g., lupus nephritis). Current treatments including steroids, antimalarials, and immunosuppressive drugs have significant side effects. Activated protein C is a natural protein with anticoagulant and immunomodulatory effects, and its recombinant version has been approved by the U.S. Food and Drug Administration to treat severe sepsis. Given the similarities between overshooting immune activation in sepsis and autoimmunity, we hypothesized that recombinant activated protein C would also suppress SLE and lupus nephritis. To test this concept, autoimmune female MRL-Fas(lpr) mice were injected with either vehicle or recombinant human activated protein C from week 14-18 of age. Activated protein C treatment significantly suppressed lupus nephritis as evidenced by decrease in activity index, glomerular IgG and complement C3 deposits, macrophage counts, as well as intrarenal IL-12 expression. Further, activated protein C attenuated cutaneous lupus and lung disease as compared with vehicle-treated MRL-Fas(lpr) mice. In addition, parameters of systemic autoimmunity, such as plasma cytokine levels of IL-12p40, IL-6, and CCL2/MCP-1, and numbers of B cells and plasma cells in spleen were suppressed by activated protein C. The latter was associated with lower total plasma IgM and IgG levels as well as lower titers of anti-dsDNA IgG and rheumatoid factor. Together, recombinant activated protein C suppresses the abnormal systemic immune activation in SLE of MRL-Fas(lpr) mice, which prevents subsequent kidney, lung, and skin disease. These results implicate that recombinant activated protein C might be useful for the treatment of human SLE.     

Alpha-actinin-binding antibodies in relation to systemic lupus erythematosus and lupus nephritis

This study investigated the overall clinical impact of anti-α-actinin antibodies in patients with pre-selected autoimmune diseases and in a random group of anti-nuclear antibody (ANA)-positive individuals. The relation of anti-α-actinin antibodies with lupus nephritis and anti-double-stranded DNA (anti-dsDNA) antibodies represented a particular focus for the study. Using a cross-sectional design, the presence of antibodies to α-actinin was studied in selected groups, classified according to the relevant American College of Rheumatology classification criteria for systemic lupus erythematosus (SLE) (n = 99), rheumatoid arthritis (RA) (n = 68), Wegener's granulomatosis (WG) (n = 85), and fibromyalgia (FM) (n = 29), and in a random group of ANA-positive individuals (n = 142). Renal disease was defined as (increased) proteinuria with haematuria or presence of cellular casts. Sera from SLE, RA, and Sj?gren's syndrome (SS) patients had significantly higher levels of anti-α-actinin antibodies than the other patient groups. Using the geometric mean (± 2 standard deviations) in FM patients as the upper cutoff, 20% of SLE patients, 12% of RA patients, 4% of SS patients, and none of the WG patients were positive for anti-α-actinin antibodies. Within the SLE cohort, anti-α-actinin antibody levels were higher in patients with renal flares (p = 0.02) and correlated independently with anti-dsDNA antibody levels by enzyme-linked immunosorbent assay (p < 0.007) but not with other disease features. In the random ANA group, 14 individuals had anti-α-actinin antibodies. Of these, 36% had SLE, while 64% suffered from other, mostly autoimmune, disorders. Antibodies binding to α-actinin were detected in 20% of SLE patients but were not specific for SLE. They correlate with anti-dsDNA antibody levels, implying in vitro cross-reactivity of anti-dsDNA antibodies, which may explain the observed association with renal disease in SLE.     

Alpha-actinin-binding antibodies in relation to systemic lupus erythematosus and lupus nephritis

This study investigated the overall clinical impact of anti-α-actinin antibodies in patients with pre-selected autoimmune diseases and in a random group of anti-nuclear antibody (ANA)-positive individuals. The relation of anti-α-actinin antibodies with lupus nephritis and anti-double-stranded DNA (anti-dsDNA) antibodies represented a particular focus for the study. Using a cross-sectional design, the presence of antibodies to α-actinin was studied in selected groups, classified according to the relevant American College of Rheumatology classification criteria for systemic lupus erythematosus (SLE) (n = 99), rheumatoid arthritis (RA) (n = 68), Wegener's granulomatosis (WG) (n = 85), and fibromyalgia (FM) (n = 29), and in a random group of ANA-positive individuals (n = 142). Renal disease was defined as (increased) proteinuria with haematuria or presence of cellular casts. Sera from SLE, RA, and Sjøgren's syndrome (SS) patients had significantly higher levels of anti-α-actinin antibodies than the other patient groups. Using the geometric mean (± 2 standard deviations) in FM patients as the upper cutoff, 20% of SLE patients, 12% of RA patients, 4% of SS patients, and none of the WG patients were positive for anti-α-actinin antibodies. Within the SLE cohort, anti-α-actinin antibody levels were higher in patients with renal flares (p = 0.02) and correlated independently with anti-dsDNA antibody levels by enzyme-linked immunosorbent assay (p < 0.007) but not with other disease features. In the random ANA group, 14 individuals had anti-α-actinin antibodies. Of these, 36% had SLE, while 64% suffered from other, mostly autoimmune, disorders. Antibodies binding to α-actinin were detected in 20% of SLE patients but were not specific for SLE. They correlate with anti-dsDNA antibody levels, implying in vitro cross-reactivity of anti-dsDNA antibodies, which may explain the observed association with renal disease in SLE.     

Familial paracentric inversions inv(2)(q31q35) and inv(8)(q22.3q24.13) ascertained through reproductive abnormalities

Summary Two cases of familial paracentric inversion, one in the long arm of chromosome 2 and the other in the long arm of chromosome 8, are described. The first was ascertained in a woman who was studied because of recurrent abortions. The second was ascertained in the father of a girl with the trichorhinophalangeal syndrome and an interstitial deletion in 8q. The latter is the first case in which unequal crossing over in an inversion loop can be inferred in a male carrier of a paracentric inversion. The reasons for the relatively low frequency of paracentric inversions observed and factors which affect the pregnancy outcome are discussed.     

Serum protein pattern associated with organ damage and lupus nephritis in systemic lupus erythematosus revealed by PEA immunoassay

Background

Systemic lupus erythematosus (SLE) is a remarkably heterogeneous autoimmune disease. Despite tremendous efforts, our knowledge of serum protein patterns in severe SLE phenotypes is still limited. We investigated the serum protein pattern of SLE, with special emphasis on irreversible organ damage and active lupus nephritis (LN) as assessed by renal Systemic Lupus Erythematosus Disease Activity Index.

Methods

We used proximity extension immunoassay (PEA, Proseek Multiplex, Olink) to assess the serum levels of ninety-two inflammation-related proteins in Czech patients with SLE (n = 75) and age-matched healthy control subjects (n = 23). Subgroup analysis was carried out on the basis of organ damage (with/without, 42/33) and biopsy-proven LN (with/without, 27/48; active LN, n = 13; inactive LN, n = 14).

Results

Of thirty deregulated proteins between SLE and the healthy controls (P _corr  < 0.05), the top upregulated proteins in SLE were sirtuin 2, interleukin 18 (IL18), and caspase 8 (P _corr  < 0.0006). Of these, sirtuin 2 and caspase 8 had not yet been reported with SLE. Elevated levels of IL8, CCL2/MCP1, CCL11, and MMP10 (P _corr  < 0.05) were detected in patients with organ damage for which the serum levels of CCL11 and MMP10 were particularly informative in organ damage prediction. Comparing patients based on LN, elevated levels of CSF1, sIL15RA, sCD40, sCX3CL1, caspase 8, sIL18R1, bNGF, and GDNF (P _corr  < 0.05) were detected in active LN. Except GDNF, all LN-associated markers showed usefulness in prediction of active renal disease.

Conclusions

This highly sensitive PEA analysis identified the serum pattern of SLE, organ damage, and active LN, with many novel candidate proteins detected. Their exact role and suitability as biomarkers in SLE deserve further investigation.

     

Anti-C1q antibodies antedate patent active glomerulonephritis in patients with systemic lupus erythematosus

Introduction

Autoantibodies against C1q correlate with lupus nephritis. We compared titers of anti-C1q and anti-dsDNA in 70 systemic lupus erythematosus patients with (n = 15) or without (n = 55) subsequent biopsy-proven lupus nephritis.

Methods

The 15 patients with subsequent lupus nephritis had anti-C1q assays during clinical flares (mean Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), 10.0 ± 4.7; range, 3 to 22) before the diagnosis of lupus nephritis (median, 24 months; range 3 to 192). Among the 55 others, 33 patients had active lupus (mean SLEDAI, 10.3 ± 6.2; range, 4 to 30) without renal disease during follow-up (median 13 years; range 2 to 17 years) and 22 had inactive lupus (mean SLEDAI, 0; range, 0 to 3).

Results

Anti-C1q titers were elevated in 15/15 (100%) patients who subsequently developed nephritis (class IV, n = 14; class V, n = 1) and in 15/33 (45%) patients without renal disease (P < 0.001). The median anti-C1q titer differed significantly between the groups (P = 0.003). Anti-C1q titers were persistently positive at the time of glomerulonephritis diagnosis in 70% (7/10) of patients, with no difference in titers compared with pre-nephritis values (median, 147 U/ml; interquartile range (IQR), 69 to 213 versus 116 U/ml; 50 to 284, respectively). Titers decreased after 6 months'' treatment with immunosuppressive drugs and corticosteroids (median, 76 U/ml; IQR, 33 to 106) but remained above normal in 6/8 (75%) patients. Anti-dsDNA antibodies were increased in 14/15 (93.3%) patients with subsequent nephritis and 24/33 (72.7%) patients without nephritis (P = ns). Anti-C1q did not correlate with anti-dsDNA or the SLEDAI in either group.

Conclusions

Anti-C1q elevation had 50% positive predictive value (15/30) and 100% (18/18) negative predictive value for subsequent class IV or V lupus nephritis.     

Complement receptor 1 (CD35) on human reticulocytes: normal expression in systemic lupus erythematosus and HIV-infected patients.

The low levels of complement receptor 1 (CR1) on erythrocytes in autoimmune diseases and AIDS may be due to accelerated loss in the circulation, or to a diminished expression of CR1 on the red cell lineage. Therefore, we analyzed the expression of CR1 on reticulocytes (R) vs erythrocytes (E). Healthy subjects had a significant higher CR1 number per cell on R (919 +/- 99 CR1/cell) than on E (279 +/- 30 CR1/cell, n = 23), which corresponded to a 3. 5- +/- 1.3-fold loss of CR1. This intravascular loss was confirmed by FACS analysis, which showed that all R expressed CR1, whereas a large fraction of E was negative. The systemic lupus erythematosus (SLE), HIV-infected, and cold hemolytic Ab disease (CHAD) patients had a CR1 number on R identical to the healthy subjects, contrasting with a lower CR1 on their E. The data indicated a significantly higher loss of CR1 in the three diseases, i.e., 7.0- +/- 3.8-, 6.1- +/- 2.9-, and 9.6- +/- 5.6-fold, respectively. The intravascular loss was best exemplified in a patient with factor I deficiency whose CR1 dropped from 520 CR1/R to 28 CR1/E, i.e., 18.6-fold loss. In one SLE patient and in the factor I-deficient patient, the FACS data were consistent with a loss of CR1 already on some R. In conclusion, CR1 is lost progressively from normal E during in vivo aging so that old E are almost devoid of CR1. The low CR1 of RBC in autoimmune diseases and HIV-infection is due to a loss occurring in the circulation by an active process that remains to be defined.     

Defective production of interleukin 1 and interleukin 2 in patients with systemic lupus erythematosus (SLE)

The effects of local antigenic exposure on the responsiveness of systemic T cells were evaluated after C3H mice were given drinking water containing 6% bovine serum albumin (BSA) for 10 days and challenged sc with 1.0 mg BSA in adjuvant 28 days after the initiation of antigen feeding. During the first 28 days, no evidence of in vitro antigen-induced proliferation [( 3H]thymidine incorporation) was detected in whole lymphocyte populations from the peripheral lymph nodes (PLN), spleen, or mesenteric nodes. In contrast, PLN cells treated with anti-Lyt-1 plus complement (C) had a significant proliferative response only if the cells were obtained during the first 6 days of antigen ingestion. Lymphoid cells from the same animals, treated with anti-Lyt-2 and C, did not respond to antigen. Two or 4 days after the injection, given on day 28, whole PLN cell populations from antigen-fed mice showed proliferation. No response was observed with PLN cells obtained 8 days after injection. Shortening the interval between the initiation of feeding and parenteral challenge partially restored proliferative responses detected 8 days after injection. Cultures prepared 4 days after simultaneous oral and parenteral antigenic exposure showed proliferation equal to or greater than cultures from mice that received only the injection. These data show that systemic T cell responsiveness is not eliminated by ingestion of soluble antigen, but rather is modulated in a manner previously detected in the humoral immune system.     

Chromosome aberrations in F1 (NZBxNZW) mice--an experimental model of systemic lupus erythematosus

Spontaneous and mitomycin-C-induced chromosomal aberration level was studied in bone marrow cells of mice F1 (NZBXNZW), an experimental model of systemic lupus erythematosus and in C57BL/6J mice. The chromosome instability level is found to correspond to the degree of the autoimmune process: F1 (NZBXNZW) mice with the highest titer of autoantibodies to DNA has higher chromosomal mutability.     

Th1/Th2 cytokine profile in childhood-onset systemic lupus erythematosus

ObjectiveTo determine the serum levels of Th1 (IL-12, IFN-γ,TNF-α) and Th2 (IL-5, IL-6 and IL-10) cytokines in childhood-onset SLE, first-degree relatives and healthy controls. To elucidate their association with disease activity, laboratory and treatment features.MethodsWe included 60 consecutive childhood-onset SLE patients [median age 18 years (range 10–37)], 64 first-degree relatives [median 40 (range 28–52)] and 57 healthy [median age 19 years (range 6–30 years)] controls. Controls were age and sex-matched to SLE patients. SLE patients were assessed for clinical and laboratory SLE manifestations, disease activity (SLEDAI), damage (SDI) and current drug exposures. Mood and anxiety disorders were determined through Becks Depression (BDI) and Anxiety Inventory (BAI). Th1 (IL-12, IFN-γ,TNF-α) and Th2 (IL-5, IL-6 and IL-10) cytokines levels were measured by ELISA and compared by non-parametric tests.ResultsSerum TNF-α (p = 0.004), IL-6 (p = 0.007) and IL-10 (p = 0.03) levels were increased in childhood-onset SLE patients when compared to first-degree relatives and healthy controls. TNF-α levels were significantly increased in patients with active disease (p = 0.014) and correlated directly with SLEDAI scores (r = 0.39; p = 0.002). IL-12 (p = 0.042) and TNF-α (p = 0.009) levels were significantly increased in patients with nephritis and TNF-α in patients with depression (p = 0.001). No association between cytokine levels and SDI scores or medication was observed.ConclusionTh1 cytokines may play a role in the pathogenesis of neuropsychiatric and renal manifestations in childhood-onset SLE. The correlation with SLEDAI suggests that TNF-α may be a useful biomarker for disease activity in childhood-onset SLE, however longitudinal studies are necessary to determine if increase of this cytokine may predict flares in childhood-onset SLE.     

The role of interleukin-10 in autoimmune disease: systemic lupus erythematosus (SLE) and multiple sclerosis (MS)

Interleukin-10 (IL-10) is an immunoregulatory cytokine that plays a crucial role in inflammatory and immune reactions. It has potent anti-inflammatory and immunosuppressive activities on myeloid cell functions which forms a solid basis for its use in acute and chronic inflammatory diseases. Here, we discuss the role of IL-10 in autoimmune diseases and examine its beneficial effects in cellular-based autoimmune diseases such as multiple sclerosis (MS) or its involvement in humoral-based autoimmune diseases such as systemic lupus erythematosus (SLE). Inhibition of the immune stimulatory activities of IL-10 may provide novel approaches in the treatment of humoral autoimmune diseases, infectious diseases and cancer.     

A conserved anti-DNA antibody idiotype associated with nephritis in murine and human systemic lupus erythematosus

In order to identify unique structural features of pathogenic autoantibodies to DNA in SLE, a murine anti-anti-DNA (anti-Id) mAb (mAb 1C7) was produced in response to immunization of lupus mice with a syngeneic anti-DNA mAb (mAb 3E10). Immunization of lupus mice with mAb 3E10 inhibited production of native anti-DNA antibodies, suppressed development of lupus kidney disease (nephritis), and induced production of anti-anti-DNA (anti-Id) antibodies. mAb 1C7 bound F(ab')2 fragments of mAb 3E10, and it bound other murine anti-DNA mAb, but not murine mAb or polyclonal serum antibodies unreactive with DNA. Moreover, binding of mAb 1C7 anti-Id to mAb 3E10 was inhibited by DNA, suggesting anti-Id binding within or near the binding site for DNA. Furthermore, mAb 1C7 bound serum IgG immunoglobulins from 9/12 patients with lupus nephritis and serum anti-DNA antibodies compared to only 3/12 SLE patients with comparable serum levels of anti-DNA antibodies, but without nephritis (p = 0.04), and only 1/53 SLE patients without serum anti-DNA antibodies, 0/49 patients with rheumatoid arthritis, and 1/47 healthy subjects (p less than 0.001). These results provide evidence that mAb 1C7 identifies a conserved Id associated with anti-DNA antibodies in murine and human SLE and may be useful as a structural probe to characterize pathogenic anti-DNA antibodies in SLE.     

Genetic linkage and transmission disequilibrium of marker haplotypes at chromosome 1q41 in human systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies to a wide range of self-antigens. Recent genome screens have implicated numerous chromosomal regions as potential SLE susceptibility loci. Among these, the 1q41 locus is of particular interest, because evidence for linkage has been found in several independent SLE family collections. Additionally, the 1q41 locus appears to be syntenic with a susceptibility interval identified in the NZM2410 mouse model for SLE. Here, we report the results of genotyping of 11 microsatellite markers within the 1q41 region in 210 SLE sibpair and 122 SLE trio families. These data confirm the modest evidence for linkage at 1q41 in our family collection (LOD = 1.21 at marker D1S2616). Evidence for significant linkage disequilibrium in this interval was also found. Multiple markers in the region exhibit transmission disequilibrium, with the peak single marker multiallelic linkage disequilibrium noted at D1S490 (pedigree disequilibrium test [PDT] global P value = 0.0091). Two- and three-marker haplotypes from the 1q41 region similarly showed strong transmission distortion in the collection of 332 SLE families. The finding of linkage together with significant transmission disequilibrium provides strong evidence for a susceptibility locus at 1q41 in human SLE.     

PCR-RFLP genotyping of C1q mutations and single nucleotide polymorphisms in Malaysian patients with systemic lupus erythematosus

Five types of known mutations within the C1q gene [located at C1qA-Gln186 (C >T), C1qB-Gly15 (G >A), C1qB-Arg150 (C >T), C1qC-Gly6 (G >A), and C1qC-Arg41 (C >T)] and two SNPs located at C1qA-Gly70 (G/A) and C1qC-Pro14 (T/C) were screened in a multiracial Malaysian population. One hundred thirty patients with systemic lupus erythematosus (SLE) and 130 matched healthy control subjects were genotyped using PCR-RFLP methods. We found no occurrence of the five types of mutations in either the homozygous or heterozygous form among the 260 samples studied. Statistical analysis also revealed that there were no significant associations observed in the genotype distributions and allele frequencies among the patients with SLE and healthy control subjects with both C1qA-Gly70 (G/A) and C1qC-Pro14 (T/C) SNPs. Overall, C1q deficiency was not proven as a primary causative genetic predisposition factor for SLE in the Malaysian population.     

Post-mortem studies on two patients with 1-2 band cytogenetic deletions: 10q26----qter and r(9)(p24q34)

Two severely retarded males with unbalanced karyotypes Case 1--(45,XY,--10,--15, + der(10)t,(10; 15) (q26; q13) and Case 2--46,XY,r(9) (p24q34) died at the ages of 24 and 38 years respectively. At post-mortem, a post-ductal coarctation of the aorta and a lump kidney were found in the first case. Brain pathology was normal in the first case and apart from a smaller size, was structurally normal in the second case.     

Essential fatty acids in diabetes and systemic lupus erythematosus (SLE) patients

Sera obtained from normal subjects and juvenile-onset diabetes (JD) and systemic lupus erythromatus (SLE) patients were examined for free fatty acid composition and 6-keto-PGF1 alpha content. In addition, prostaglandins in urine samples from normal and diabetic individuals were separated by HPLC, and 6-keto-PGF1 alpha levels were monitored by RIA. Arachidonate (20:4) content in diabetic and SLE individuals were significantly lower than that of controls. Urine from diabetic individuals showed decreased levels of 6-keto-PG F1 alpha. The study also indicated that RIA measurements on crude biological samples may yield erroneous data due to immune cross reactivity with other compounds.     

Localization and replication of the systemic lupus erythematosus linkage signal at 4p16: interaction with 2p11, 12q24 and 19q13 in European Americans

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by both population and phenotypic heterogeneity. Our group previously identified linkage to SLE at 4p16 in European Americans (EA). In the present study we replicate this linkage effect in a new cohort of 76 EA families multiplex for SLE by model-free linkage analysis. Using densely spaced microsatellite markers in the linkage region, we have localized the potential SLE susceptibility gene(s) to be telomeric to the marker D4S2928 by haplotype construction. In addition, marker D4S394 showed marginal evidence of linkage disequilibrium with the putative disease locus by the transmission disequilibrium test and significant evidence of association using a family-based association approach as implemented in the program ASSOC. We also performed both two-point and multipoint model-based analyses to characterize the genetic model of the potential SLE susceptibility gene(s), and the lod scores both maximized under a recessive model with penetrances of 0.8. Finally, we performed a genome-wide scan of the total 153 EA pedigrees and evaluated the possibility of interaction between linkage signals at 4p16 and other regions in the genome. Fourteen regions on 11 chromosomes (1q24, 1q42, 2p11, 2q32, 3p14.2, 4p16, 5p15, 7p21, 8p22, 10q22, 12p11, 12q24, 14q12, 19q13) showed evidence of linkage, among which, signals at 2p11, 12q24 and 19q13 also showed evidence of interaction with that at 4p16. These results provide important additional information about the SLE linkage effect at 4p16 and offer a unique approach to uncovering susceptibility loci involved in complex human diseases.