Rheumatoid factor induction in murine models of liver injury.

Alcoholic liver disease and hepatitis C are associated with the production of autoantibodies such as rheumatoid factors (RF), which bind to IgG and can aid in host defence, but are also associated with pathological conditions such as rheumatoid arthritis. Because little is known about the role of RF in liver disease, we characterized the RF production that either occurred spontaneously in response to alcohol consumption or was induced by injection of an Escherichia coli glycolipoprotein in C57Bl/6 mice. Whereas severe liver damage was induced by carbon tetrachloride (CCl(4)), minimal damage was caused by chronic alcohol consumption. Liver damage was monitored by measurements of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Circulating RF was induced in response to chronic alcohol consumption; the latter probably involved Toll-like receptor ligation. In contrast, CCl(4)-induced damage was not associated with RF induction. However, concurrent treatment with an E. coli glycolipoprotein macromolecule that induced RF, protected against CCL(4)-induced liver damage as measured by a highly significant decrease (P = 0.008) at 4 weeks in AST and ALT. RF induced by E. coli glycolipoprotein correlated with 'protection' from liver damage, indicating that the RF autoimmune response does not necessarily exacerbate liver disease.

Agonistic antibodies to Fas induce a breach in the endothelial lining of the liver and a breakdown in B cell tolerance.

Liver disease can be associated with a breakdown in self-tolerance and the production of autoantibodies such as rheumatoid factors (RF), which bind to IgG. Here we investigated whether primary, non-infectious liver damage was sufficient to induce autoantibody production. We established a model of targeted liver damage induced by weekly sublethal injections of pro-apoptotic anti-Fas (CD95) antibodies. Liver damage, monitored by measurements of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, was minimal 1 week after anti-Fas injection. However, the sublethal Fas stimulation was sufficient to trigger significant haemorrhage in the liver, as assessed by Evans Blue dye leakage into the organ 5 h after anti-Fas antibody injection. We observed an induction of RF in response to the weekly injections of sublethal anti-Fas antibodies but not of isotype control antibodies, indicating a breakdown of self-tolerance induced by Fas engagement. RF induction was unlikely to be due to direct activation of B cells, as splenocytes stimulated with anti-Fas antibodies in vitro did not produce RF. These studies show that sublethal damage to the liver by Fas engagement leads to liver haemorrhage and is sufficient to trigger the breakdown of self-tolerance.

Elevated levels of IgM and IgA antibodies to Proteus mirabilis and IgM antibodies to Escherichia coli are associated with early rheumatoid factor (RF)-positive rheumatoid arthritis.

OBJECTIVE: Antibodies to Proteus mirabilis were previously detected in patients with established rheumatoid arthritis (RA). We examined the prevalence of antibodies to P. mirabilis and their associations with RA in early synovitis patients. METHODS: Two hundred and forty-six patients with inflammatory arthritis for less than 1 yr were prospectively evaluated for 1 yr. Of these patients, 30% had rheumatoid factor (RF)-positive RA, 16% RF-negative RA, 17% a spondyloarthropathy and 37% undifferentiated arthritis. Serum antibodies to P. mirabilis, Escherichia coli and other potentially arthritogenic organisms (Chlamydia, Salmonella, Shigella, Campylobacter, Yersinia and parvovirus B19) and for antibodies specific for immunoglobulin (Ig) G damaged with advanced glycation end-products (anti-IgG-AGE) were measured. RESULTS: IgM and IgA anti-Proteus antibodies were significantly higher in patients with RF-positive RA compared with all other patient groups (P < 0.0005 and P < 0.005). Anti-P. mirabilis IgG, and IgG, IgA, and IgM antibodies to other potentially arthritogenic pathogens did not differ in the patient groups. IgM antibodies to E. coli were elevated in RF-positive RA patients. Anti-P. mirabilis IgM and IgA results were not explained by false-positive reactions, because after absorption of RF there was no decrease in antibodies to Proteus in 10 of 12 patients. Proteus and E. coli antibodies were highest in patients positive for both RF and anti-IgG-AGE antibodies (P<0.001). Patients with erosions tended to have higher IgA anti-Proteus titres, but no association with the shared HLA epitope or treatment was detected. CONCLUSION: Anti-P. mirabilis IgM and IgA and anti-E. coli IgM antibody elevations are associated with early seropositive RA and the presence of anti-IgG-AGE antibodies. The role that P. mirabilis or E. coli plays in early RF-positive RA requires further investigation.

The genotype of mice influences the autoimmune response to spliceosome proteins induced by cytomegalovirus gB immunization.

In previous studies we have established a link between cytomegalovirus (CMV) infection and an autoimmune response to the U1-70 k protein of the spliceosome in man. This autoimmune response, generally referred to as the anti-RNP (ribonucleoprotein) antibodies, is observed in about 30% of patients with systemic lupus erythematosus (SLE). We have also found that the CMV glycoprotein B (CMV gB) when expressed in a adenovirus vector (Ad) could induce a significant anti-U1-70 k antibody response in several strains of mice, such as C3H, MRL and BALB/c. In the present study we examined the autoimmune response induced by immunization with Ad-gB in A/J and C57BL/6 (B6) mice and determined whether there was any autoimmune phenotype similar to that observed in patients with SLE. Thus groups of A/J and B6 mice were immunized with Ad/gB or with Ad alone and then observed for possible skin or kidney disease. In addition the autoantibody response to the spliceosome was measured, and the target antigens identified by immunoblot techniques. All of the A/J mice mounted a very high IgG response primarily to the U1-70 k protein of the spliceosome, with evidence of a rapid spreading of the autoantibody response to other components of the complex. In contrast, B6 mice mounted only a very low titre autoantibody response and failed to show signs or symptoms of autoimmunity. The A/J but not the B6 mice were found to have deposits of IgG in their kidneys, which were consistent with abnormal levels of blood urea nitrogen in the A/J but not B6 mice. This study demonstrates the importance of the genetic background in the susceptibility to autoimmunity.

Autoimmune response to U1 small nuclear ribonucleoprotein (U1 snRNP) associated with cytomegalovirus infection.

The induction of autoantibodies to U1 small nuclear ribonucleoprotein (U1 snRNP) complexes is not well understood. We present evidence that healthy individuals with cytomegalovirus (CMV) infection have an increased frequency and quantity of antibodies to ribonucleoprotein, directed primarily against the U1-70k protein. A significant association between the presence of antibodies to CMV and antibodies to the total RNP targeted by the immune response to the spliceosome (to both the Sm and RNP; Sm/RNP) was found for patients with systemic lupus erythematosus (SLE) but not those with mixed connective-tissue disease. CMV thus may play a role in inducing autoimmune responses in a subset of patients with systemic lupus erythematosus.

An autoantibody targeting glycated IgG is associated with elevated serum immune complexes in rheumatoid arthritis (RA).

Advanced glycation end-products (AGE) play a role in diabetes complications and in RA. An autoantibody to IgG-AGE has been shown to correlate with RA disease activity. Thus we sought to analyse serum immune complexes (IC) and AGE-modified proteins in Caucasians and North American Indians to see if the presence of anti-IgG-AGE influenced their composition. Polyethylene glycol precipitation of IC from the serum of anti-IgG-AGE-positive or -negative RA patients, and healthy and diabetic controls were examined. Concentrations of circulating IC were highest in anti-IgG-AGE+ RA patients, followed by anti-IgG-AGE- RA patients, which were greater than healthy controls. IC amounts in the Ojibwe were consistently higher than in Caucasians. Affinity purification of AGE-modified proteins from IC and immunoblotting with antibodies against Ig gamma and mu heavy chains, kappa and lambda light chains, and AGE Nepsilon(carboxymethyl)lysine and imidazolone yielded similar results: anti-AGE+ RA patients had elevated levels relative to those without the autoantibody. Levels in both RA groups were higher than in controls. Glycated albumin amounts followed a similar distribution, but were not influenced by the presence of anti-AGE antibodies. A heavily glycated kappa-chain was present primarily in IC from anti-IgG-AGE+ patients. These studies indicate that anti-AGE antibodies have a direct impact on the accumulation of IgG-AGE but not glycated albumin, and may block the normal clearance of IgG-AGE through AGE receptors.

Association between IgM response to IgG damaged by glyoxidation and disease activity in rheumatoid arthritis.

OBJECTIVE: To determine the association of serum IgG advanced glycation endproducts (AGE) and IgM anti-IgG-AGE antibodies with clinical measurements of rheumatoid arthritis (RA) disease activity. METHODS: The study group consisted of 62 patients with RA and 16 control patients with osteoarthritis. Patient derived variables included perceived disease activity (10 cm visual analog scale, VAS) and Health Assessment Questionnaire (HAQ) results. Clinical measures of RA activity consisted of tender and swollen joint counts and a physician evaluation of disease activity (by VAS) as well as history of nodules, bone erosions, Sjogren's syndrome, and vasculitis documented by chart review. Patient sera were evaluated for glucose, glycosylated hemoglobin, and presence of RF, IgG-AGE and IgM anti-IgG-AGE. The nitroblue tetrazolium colorimetric and aminophenyl boronic acid methods were used for measurement of IgG-AGE, along with an ELISA for measurement of IgM anti-IgG-AGE. RESULTS: Significant correlations were found between the presence of IgM anti-IgG-AGE and clinical measurements of swollen joint count and physician VAS. CONCLUSION: IgM anti-IgG-AGE appears to be associated with clinical measurements of RA activity and represents a new marker of more active disease in RA.

Recombinant cytomegalovirus glycoprotein gB (UL55) induces an autoantibody response to the U1-70 kDa small nuclear ribonucleoprotein.

Human cytomegalovirus (CMV) infection can be life threatening in the immune compromised and is associated with congenital defects and / or mental retardation in the neonate. The demonstrated association between CMV infection and rheumatoid factor (RF) raised the possibility of an induction of an autoimmune response upon vaccination with a candidate CMV vaccine, glycoprotein gB (UL55). The antibody responses generated after injections of an adenovirus-gB construct (Ad-gB) were studied in autoimmune-prone (MRL/mpj) and normal (BALB.k, C3H, and BALB/c) mice. Enzyme-linked immunosorbent assay and immunoblot analyses were done to identify the autoantibodies produced following immunization. Immunization with Ad-gB induced a significant IgG anti-viral response in all strains tested (p < 0.0001) compared to phosphate-buffered saline or HeLa controls. Ad-gB induced a significant IgG autoantibody response (p > 0.005) to the U1-70 kDa spliceosome protein in both autoimmune and normal strains whereas immunization with recombinant human La/SS-B did not. Autoantibodies to U1-70 kDa are part of the anti-ribonucleoprotein response seen in systemic lupus erythematosus and mixed connective tissue disease. Low levels of IgG RF and anti-double-stranded DNA antibodies were also induced. This study raises concern that immunization with CMV gB in individuals genetically predisposed to autoimmunity could trigger the development or acceleration of an autoimmune disease.

A measure of limited joint motion and deformity correlates with HLA-DRB1 and DQB1 alleles in patients with rheumatoid arthritis.

OBJECTIVE: To assess factors associated with a poor outcome in rheumatoid arthritis (RA), a measure was developed of limited joint motion and deformity, a deformity index (DI), and correlated biochemical and genetic variables with the magnitude of the DI. METHODS: Forty patients were evaluated in a cross sectional study. Clinical measures included the DI and Health Assessment Questionnaire, and disease variables included the erythrocyte sedimentation rate, C reactive protein, rheumatoid factor, and HLA-DRB1 and DQB1 alleles. RESULTS: Significant correlations were noted between increasing DI and duration of RA and concentration of C reactive protein. Patients with a DQB1*301 allele or DR4 allele had a higher DI than those without, and a positive trend was noted between increasing DI and dose of DRB1 RA susceptibility alleles. The trend was lost when a non-linear regression technique was used to remove the effect attributable to C reactive protein, suggesting an interrelation between persistent inflammation and genetics in determining total joint damage. CONCLUSIONS: The DI may be useful to study interactions between genetic and inflammatory processes in rheumatoid disease progression.

Systemic lupus erythematosus, a disease associated with low levels of clusterin/apoJ, an antiinflammatory protein.

OBJECTIVE: To measure the serum levels of clusterin, an antiinflammatory protein, which binds and inactivates complement, in patients with systemic lupus erythematosus (SLE) to determine whether the levels correlate with disease. METHODS: The levels of serum clusterin were measured by ELISA in 80 patients with SLE (76 female, 4 male). Clinical and serological information was gathered on 115 visits. Overall disease activity scores were determined using the Systemic Lupus Activity Measure-Revised. RESULTS: Serum clusterin levels were significantly decreased in patients with SLE and correlated inversely with disease activity (p < 0.00001). Low clusterin levels were significantly associated with skin ulcers (p < 0.0001), loss of hair (p = 0.002), proteinuria (p = 0.018), low platelet count (p = 0.03), and arthritis (p < 0.0001). The clusterin levels did not correlate with either systemic complement consumption, as measured by C3 or C4, or with prednisone use. CONCLUSION: A highly significant correlation was observed between low levels of serum clusterin and a number of SLE disease features. This deficiency of clusterin could directly or indirectly affect the disease process. Individuals lacking sufficient amounts of clusterin systemically likely have poor control of antibody mediated inflammation at sites of apoptosis where autoantigens are exposed.

Advanced glycation endproducts (AGE) on IgG, a target for circulating antibodies in North American Indians with rheumatoid arthritis (RA).

Several tribes of North American Indians are known to have poor glucose control and are at a high risk of developing type 2 diabetes. Similarly some tribes also exhibit RA at a high frequency. We have recently determined that a subset of Caucasian patients with RA mount an immune response to IgG modified with advanced glycation endproducts (AGE). The AGE modifications on IgG in vivo include N(epsilon)-(carboxymethyl) lysine, imidazolone and pentosidine. The presence of IgG-AGE and the antibody response to the IgG-AGE in the Ojibwe tribe of First Nations native Indians where both NIDDM and RA are prevalent was investigated. AGE modified IgG and albumin were determined using a modified nitroblue tetrazolium assay. Rheumatoid factors (RFs) and IgM and IgA anti-IgG-AGE were detected by ELISA. Of the 108 individuals tested, 21 had RA only, 3 had both RA and type 2 diabetes, 30 had type 2 diabetes only and 51 had no diagnosed disease. AGE modified IgG was significantly elevated in the RA group compared to the diabetic group. IgM and IgA RFs were detected in 83% and 50% of the RA patients, compared to 31-37% and 7-10% of the diabetics or normal individuals. IgM anti-IgG-AGE was detected in 54% of the RA patients, in contrast to 7-14% in the diabetics or normal individuals. IgA anti-IgG-AGE was detected in 42% of the RA patients and only 7 to 8% of the NIDDM or normal individuals. The IgM or IgA anti-IgG-AGE antibodies likely contribute to the accumulation of IgG-AGE, possibly through blocked clearance through AGE receptors. A trend towards more severe disease was seen in those Ojibwe RA patients with circulating anti-AGE antibodies. Non-enzymatic glycation may be an important pathogenic link in the RA seen in North American Indians.

A new antibody in rheumatoid arthritis targeting glycated IgG: IgM anti-IgG-AGE.

Hyperglycaemia and/or oxidative stress can cause IgG to be modified by advanced glycation end products (AGE). Three patients with aggressive rheumatoid arthritis (RA) and vasculitis are described who have high titres of IgM antibodies against AGE-modified IgG (IgM anti-IgG-AGE). Diabetics and randomly selected patients with rheumatic diseases, including 50 additional RA patients, were tested for IgM and IgA anti-IgG-AGE by ELISA. AGE-modified proteins were detected using the nitroblue tetrazolium (NBT) colorimetric method. The presence of Nepsilon (carboxymethyl) lysine, an AGE modification, was detected on IgG-AGE by immunoblotting. A total of 20/41 (49%) rheumatoid factor (RF)-positive RA patients tested had IgM anti-IgG-AGE antibodies, 4/12 (33%) RF-positive systemic lupus erythematosus (SLE) patients, 3/5 RF-positive patients with primary Sjogren's syndrome (SS), and 3/5 RF-positive diabetics. All patients with RF-negative RA, SLE, SS, osteoarthritis (24), spondyloarthritis (15), adult-onset Still's disease (8), diabetes (25) and healthy controls (20) were anti-IgG-AGE negative. RF and IgM anti-IgG-AGE appeared to be a linked response. The IgM anti-IgG-AGE, along with IgG-AGE, may contribute to the pathogenesis of RA.

Differential clearance of glycoforms of IgG in normal and autoimmune-prone mice.

In order to understand better the origins of the elevated levels of the glycoform of IgG that lacks galactose on both arms of the oligosaccharide chain (G0%) located in the Fc, which occurs in man and mouse with age, and in particular in autoimmune disease, we investigated the clearance of two glycosylated forms of IgG2a and IgG1 in normal (BALB/c) and autoimmune-prone (MRL/1pr, MRL/+, and non-obese diabetic (NOD)) mice. To investigate the possibility of different rates of catabolism, enzymatically generated glycoforms of monomeric IgG1 and IgG2a (fully glycosylated or G0%), were iodinated and injected into the tail vein of the mice. We found that the G0% IgG2a remained in circulation significantly longer than the fully glycosylated variants, in all of the mouse strains tested. In contrast, the two forms of IgG1 had similar kinetics in all the autoimmune-prone mice, whereas in BALB/c, there was a longer half-life (t1/2) for G0% IgG1. These data suggest that there may be differences in the ability of the IgG glycoforms to bind to the Fc gamma receptors, in particular Fc gamma RI. The clearance rates were found to vary among the strains studied, with MRL/1pr having the fastest catabolic rates for all glycoforms and IgG subclasses tested. This appeared to be due to the presence of circulating IgG and IgM rheumatoid factors (RF). There were significantly increased frequencies and titres for both IgM and IgG RF in MRL/1pr mice compared with the other strains. In contrast, interferon-gamma, known to induce the Fc gamma RI, was found to be similar in the sera, in all of the strains of mice examined. These results suggest that RF probably play an important biological function in the MRL/1pr mice and aid in the clearance of circulating IgG. Our study shows that the state of glycosylation of IgG affects the t1/2 in vivo, and that by removing the terminal sugars (sialic acid and galactose), the antibody (IgG2a) will remain in circulation significantly longer. These observations may thus provide a partial explanation for the increase in relative percentage of this glycoform that occurs with age.

Rheumatic disease patients, prone to Sjögren's syndrome and/or lymphoma, mount an antibody response to BHRF1, the Epstein-Barr viral homologue of BCL-2.

The IgG response to Epstein-Barr virus (EBV) early antigens [BHRF1 (p 17.1), the viral homologue of bcl-2, and BMRF1 (p50.10), a DNA binding protein] was measured in patients with rheumatic disease to see whether there was any association with lymphoma. Patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), rheumatic disease patients with lymphoma, patients with lymphoma who did not have a rheumatic disease and normal individuals were tested for the presence of anti-EA peptide antibodies by ELISA. Whereas antibodies to early EBV peptides were detected only in one normal individual, patients with rheumatic diseases, especially those with either SS and/or lymphoma, had a much higher frequency of antibody detection. Antibodies to BMRF1 p50.10 were found in 7-50% of patients, and to BHRF1 p17.1 in 4-27%, depending on the group studied. Patients with lymphoma lacking a rheumatic disease had a 2-fold lower frequency of anti-BHRF1 antibodies, compared to the lymphoma plus rheumatic disease group. The increased immune response to the EBV EA proteins in the rheumatic diseases probably reflects the presence of reactivated virus, and the BHRF1 protein (the viral homologue to bcl-2) could, via inhibiting apoptosis, contribute to the lymphoproliferative nature of these diseases.

Herpes viruses in multicase families with rheumatoid arthritis.

OBJECTIVE: To investigate the rate and extent of infection by Epstein-Barr virus (EBV), cytomegalovirus, and herpesvirus 6 in families (affected and nonaffected members) with multiple cases of rheumatoid arthritis (RA). METHODS: Viral DNA was detected by polymerase chain amplification in cells from saliva and peripheral blood. Human leukocyte antigen pedigrees were characterized. RESULTS: Viral DNA, particularly EBV, was detected in increased frequency (p = 0.029) in the patients with RA compare to their nonaffected relatives. CONCLUSION: We suggest that in RA multicase families, increased frequency of viral infection is likely a consequence of the disease state and/or due to gene(s) as yet unidentified.

Rheumatoid factor avidity in patients with rheumatoid arthritis: identification of pathogenic RFs which correlate with disease parameters and with the gal(0) glycoform of IgG.

The standard ELISA for measuring rheumatoid factor (RF) binding was modified by treatment after the RF-Fc interaction with 2 M guanidine, which allowed a measurement of the avidity of the interaction. Incubation with 4 M guanidine eliminated RF binding. There was a direct correlation (r = 0.99) between the avidity as measured by the modified guanidine ELISA, and the dissociation constant for monoclonal RFs, as measured by competitive ELISA. Of the seropositive rheumatoid arthritis (RA) patients tested, 47% had high-avidity RFs (> or = 8% RF binding remaining after guanidine treatment). Tender joint count scores were significantly higher in the high avidity group (p = 0.05), whereas there was no significant difference in the ages, disease duration, sedimentation rate, RF titer or serum Ig levels compared to those with low-avidity RFs. Additionally 58% of those with high-avidity RFs had subcutaneous nodules, compared to 40% of the low-avidity group. A significantly higher number of nodules was present in the high-avidity RF group compared to those with low-avidity RFs (p = 0.03). Interestingly, the RF avidity was significantly higher in isolated immune complexes (IC), compared to that in circulating IgM RFs (p = 0.01). The RF avidity correlated with the presence of the glycoform of IgG lacking galactose in both circulating and IC-derived IgG (p = 0.003 and 0.009 respectively). Information about the strength of binding to Fc identifies a subgroup of IgM RFs that are likely pathological in patients with RA, as well as a specific glycoform of the target antigen.

Immunoglobulin gene usage in the human anti-pathogen response.

The human antibody response to foreign pathogens is generated to a relatively small number of target surface proteins and carbohydrates that nonetheless have an extensive array of epitopes. The study of human monoclonal antibodies to different pathogens shows that there are a diversity of mechanisms used to generate a sufficient repertoire of antibodies to combat the invading pathogens. Although many different immunoglobulin gene elements are used to construct the anti-pathogen response, some elements are used more often than would be expected if all elements were used randomly. For example, the immune response to Haemophilus influenzae polysaccharide appears to be quite narrow, being restricted primarily to a specific heavy-chain gene, 3-15, and a lambda light-chain family II member, 4A. In contrast, for the immune response to cytomegalovirus proteins, a wider group of gene elements is needed. It is also surprising that despite an investigator bias for IgG- rather than IgM-secreting immortal B cells (because of their high affinity and neutralizing abilities), 26% of light chains and 13% of heavy chains showed a very low level of somatic mutation, equivalent to an IgM molecule that has not undergone affinity maturation. Although some highly mutated IgG molecules are present in the anti-pathogen response, most of the monoclonal antibodies specific for viruses or bacteria have a level of somatic hypermutation similar to that of the adult IgM repertoire. A number of studies have shown that there are similarities in the antibody responses to pathogens and to self (autoantibodies).(ABSTRACT TRUNCATED AT 250 WORDS)