Marianna M. Newkirk

Marked differences in fine specificity and isotype usage of the anti–citrullinated protein antibody in health and disease

OBJECTIVE Anti-citrullinated protein antibodies (ACPAs) display high association with rheumatoid arthritis (RA) and are implicated in its pathogenesis. The presence of ACPAs is known to precede the onset of RA. In order to identify the features that could confer its pathogenicity, we extensively characterized this antibody response in a unique North American native population of patients with RA and their unaffected relatives. METHODS The levels of IgA, IgM, and IgG ACPAs, as well as IgM and IgA rheumatoid factor (RF), were measured in serum samples obtained from 81 patients with RA and 195 of their unaffected relatives. The isotype distribution, the fine specificity of the ACPA response, and its association with RF were compared in health and disease. RESULTS ACPA positivity was observed in 19% of the healthy relatives and approximately 91% of the patients with RA. ACPA isotype usage was strikingly lower in unaffected relatives than in patients with RA (1-2 versus 5-6 isotypes). Fine specificity studies showed that reactivity to citrullinated fibrinogen and vimentin was present in sera from patients with RA, while it was virtually absent in their unaffected relatives. Finally, the ACPA and RF responses were associated in patients with RA but were discordant in their healthy relatives. Extended analyses revealed that the presence of ACPAs was associated with RA irrespective of RF status, while the association of RF with disease relied on its interaction with ACPAs. CONCLUSION The fine specificity and isotype usage of the ACPA response are qualitatively different in health and disease. Epitope spreading and expansion of the isotype repertoire might be necessary for development of RA, and this could be facilitated by the presence of RF antibodies.

Antibodies to Porphyromonas gingivalis Are Associated with Anticitrullinated Protein Antibodies in Patients with Rheumatoid Arthritis and Their Relatives

Objective. Anticitrullinated protein antibodies (ACPA) are relatively specific for rheumatoid arthritis (RA), and predate disease. The oral pathogen Porphyromonas gingivalis may play a role in breaking immune tolerance to citrullinated antigens. We studied a cohort of patients with RA and their relatives looking for associations between anti-P. gingivalis antibodies and ACPA. Methods. Patients with RA (n = 82) and their relatives (n = 205) from a North American Native (NAN) population were studied, along with 47 NAN and 60 non-NAN controls. IgM and IgA rheumatoid factor (RF) were tested by nephelometry and ELISA. Second-generation anticyclic citrullinated peptide (anti-CCP2) isotypes and IgG anti-P. gingivalis lipopolysaccharides were tested by ELISA. HLA-DRB1 typing was performed by sequencing. Oral hygiene and smoking habits were assessed by questionnaires. Results. Autoantibody frequency in patients with RA and relatives: ACPA 91% vs 19%, respectively; IgM RF 82% vs 17%; IgA RF 48% vs 22%. Anti-P. gingivalis levels were higher in patients with RA compared to relatives and controls (p = 0.005) and higher in ACPA-positive patients with RA than in ACPA-negative patients with RA (p = 0.04) and relatives (p < 0.001), but comparable in RF-positive and RF-negative patients and relatives. Poor oral hygiene and smoking were prevalent, but with no clear association with autoantibodies. Relatives with 2 shared-epitope alleles were more likely to be ACPA-positive (OR 2.5, p = 0.02). Conclusion. In a genetically predisposed population of NAN patients with RA and their relatives, anti-P. gingivalis antibodies were associated with ACPA. These findings suggest that immune responses to P. gingivalis may be involved in breaking immune tolerance to citrullinated antigens.

Familial clustering of the serum cytokine profile in the relatives of rheumatoid arthritis patients

OBJECTIVE Rheumatoid arthritis (RA) is prevalent in North American Native populations, with a high frequency of multicase families and seropositivity in first-degree relatives. This study was undertaken to determine whether the serum cytokine profile of first-degree relatives of North American Native patients with RA differed from that of individuals with no family history of autoimmunity and whether there was an association with RA autoantibodies. METHODS North American Native patients with RA (n = 105), their first-degree relatives (n = 273), healthy North American Native controls (n = 200), and Caucasian controls (n = 150) were studied. Serum levels of 42 cytokines were tested using a multiplex laser bead assay. Rheumatoid factor (RF), anti-cyclic citrullinated peptide 2 (anti-CCP-2), monocyte chemotactic protein 1 (MCP-l), and high-sensitivity C-reactive protein (hsCRP) were tested by enzyme-linked immunosorbent assay, and HLA-DRB1 alleles by specific primers. Discriminant analysis and logistic regression classified individuals based on their cytokine profile. RESULTS The prevalence of RF (cutoff level predetermined to include 5% of Caucasian controls) and anti-CCP (cutoff level of ≥40 units) was, respectively, 88% and 81% in the RA patients, 34% and 9% in first-degree relatives, and 9% and 4% in North American Native controls; the prevalence of anti-CCP was 0% in Caucasian controls. Levels of most cytokines were highest in RA patients; 17 of 40 cytokines (43%) were significantly higher in first-degree relatives than in controls, including multiple proinflammatory cytokines. Discriminant analysis showed a notable distinction between the groups, with 85% classification accuracy. First-degree relatives had markedly higher MCP-1 and hsCRP levels than North American Native controls, but there was no consistent association with RA autoantibodies. CONCLUSION Our findings indicate that levels of multiple cytokines and hsCRP are higher in first-degree relatives of North American Native patients with RA compared to individuals from a nonautoimmune background. These data suggest that elevated baseline cytokine levels may be part of the risk profile for developing RA.

Rheumatoid factor avidity in patients with rheumatoid arthritis: Identification of pathogenic RFs which correlate with disease parameters and with the gal(O) 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 (≥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.

Restricted immunoglobulin variable region gene usage by hybridoma rheumatoid factors from patients with systemic lupus erythematosus and rheumatoid arthritis

Rheumatoid factors (RFs) are autoantibodies that are produced by approximately 75% of patients with rheumatoid arthritis (RA). Their role in pathogenesis is not well understood. In this study of 81 human hybridoma IgM antibodies derived from unstimulated peripheral blood B-cells of patients with RA and systemic lupus erythematosus (SLE), we have demonstrated that idiotypes associated with RFs derived from patients with mixed cryoglobulinemia were expressed by approximately 60% of RFs and 6% of IgM antibodies lacking RF activity. The specificity of the RFs for the Fc portion of IgG only (monospecificity) or for Fc and additional self antigens (polyreactivity) was found to correlate with the expression of specific heavy chain associated idiotypes. The VH3 associated RF idiotypes, D12 and B6, were expressed by 0/16 (0%) of monospecific RFs compared with 6/22 (27%) of polyreactive RFs. The predominant use of VH3 was verified by analysis of the expressed Ig with VH family specific anti-peptide antibodies. The light chains expressed by both populations of IgM RFs were found to be predominantly VKIII, both by detection of specific epitopes/idiotypes and V family analysis. This non-random gene usage of both the heavy and light chains suggests that there is a selective expression of V regions in the RF producing B-cells in patients with RA and SLE. We suggest that different antigen-driven, clonal selection events may occur which result in either monospecific RFs or polyreactive RFs.

Restricted variable region gene usage and possible rheumatoid factor relationship among human monoclonal antibodies specific for the AD-1 epitope on cytomegalovirus glycoprotein B☆

The nucleotide sequences of the variable region genes encoding five different human, high affinity antibodies, specific for the major neutralization determinant (AD-1) expressed by human cytomegalovirus glycoprotein B (gp58/116), have been determined. Three of the five heavy chain variable regions belonged to the small VHV-family, although they combined with a diverse set of light chains (V kappa IIIb, V lambda II and V lambda III). The other two antibodies belonged to VH-families III and IV. One of the VHV-family genes most likely originated from a previously unreported germline gene or allele, since it carries a nine nucleotide insert in framework 1. In addition, V lambda-genes showed variable homology (77-95%) to known germline sequences, while V kappa-genes showed high homology (approximately 98%) with their proposed germline origin. Despite the close homology of the V kappa IIIb-gene used to express one of the antibodies with its corresponding germline gene, the protein did not strongly express some idiotypes associated with this light chain family. There is, thus, no direct relation between the expression of these crossreactive idiotypes and the use of even modestly mutated light chains belonging to this V kappa-family, which has been implicated in the development of anti-idiotypic networks possibly inducing autoantibodies, such as rheumatoid factors.

Distinct bacterial colonization patterns of Escherichia coli subtypes associate with rheumatoid factor status in early inflammatory arthritis

OBJECTIVES The aetiology of RA is unknown; however, bacterial exposure, particularly to Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae, has been linked to disease pathogenesis. The strongest association was observed for RF(+) RA. We compare colonization patterns of these bacteria, and the anti-bacterial antibody levels in early onset RF(+) and RF(-) inflammatory arthritis. METHODS Bacteria isolated from stool and urine of early-stage RF(+) and RF(-) patients recruited to the Early Arthritis Registry were biochemically identified and genotyped. IgM and IgA anti-bacterial and RF antibodies were assessed by ELISA. RESULTS Differences in the types of colonizing pathogenic E. coli were identified. RF(+) patients were more commonly colonized with phylogenetic Group D E. coli, whereas RF(-) patients were more commonly colonized with phylogenetic Group B2 E. coli and these individuals also had lower joint scores and inflammatory markers yet higher IgA anti-E. coli antibody responses. CONCLUSIONS These studies link the type of colonizing bacteria in the gut and urine with the immune response (anti-bacterial and RF) in early-onset inflammatory arthritis and provide evidence for a role of the host-pathogen response in the aetiology of RF.

Chronic smoke exposure induces rheumatoid factor and anti‐heat shock protein 70 autoantibodies in susceptible mice and humans with lung disease

The impact of cigarette smoke (CS), a risk factor for rheumatoid arthritis (RA), on sauto‐antibody production was studied in humans and mice with and without chronic lung disease (LD). Rheumatoid factor (RF), anti‐cyclic citrullinated peptides (CCPs), and anti‐HSP70 autoantibodies were measured in several mouse strains and in cohorts of smokers and nonsmokers with and without autoimmune disease. Chronic smoking‐induced RFs in AKR/J mice, which are most susceptible to LD. RFs were identified in human smokers, preferentially in those with LD. Anti‐HSP70 auto‐antibodies were identified in CS‐exposed AKR/J mice but not in ambient air exposed AKR/J controls. Whereas inflammation could induce anti‐HSP70 IgM, smoke exposure promoted the switch to anti‐HSP70 IgG autoantibodies. Elevated anti‐CCP autoantibodies were not detected in CS‐exposed mice or smokers. AKR/J splenocytes stimulated in vitro by immune complexes (ICs) of HSP70/anti‐HSP70 antibodies produced RFs. The CD91 scavenger pathway was required as anti‐CD91 blocked the HSP70‐IC‐induced RF response. Blocking Toll‐like receptors did not influence the HSP70‐IC‐induced RFs. These studies identify both anti‐HSP70 and RFs as serological markers of smoke‐related LD in humans and mice. Identification of these autoantibodies could suggest a common environmental insult, namely CS, in a number of different disease settings.

Prevalence of Anti-Peptidylarginine Deiminase Type 4 Antibodies in Rheumatoid Arthritis and Unaffected First-degree Relatives in Indigenous North American Populations

Objective. To determine whether anti-peptidylarginine deiminase type 4 (PAD4) antibodies were present in first-degree relatives (FDR) of patients with rheumatoid arthritis (RA) in 2 indigenous North American populations with high prevalence of RA. Methods. Participants were recruited from 2 indigenous populations in Canada and the United States, including patients with RA (probands), their unaffected FDR, and healthy unrelated controls. Sera were tested for the presence of anti-PAD4 antibodies, anticyclic citrullinated peptide (anti-CCP) antibodies, and rheumatoid factor (RF). HLA-DRB1 subtyping was performed and participants were classified according to number of shared-epitope alleles present. Results. Antibodies to PAD4 were detected in 24 of 82 (29.3%) probands; 2 of 147 (1.4%) relatives; and no controls (p < 0.0001). Anti-CCP was present in 39/144 (27.1%) of the relatives, and there was no overlap between positivity for anti-CCP and PAD4 in the relatives. In RA patients, anti-PAD4 antibodies were associated with disease duration (p = 0.0082) and anti-CCP antibodies (p = 0.008), but not smoking or shared-epitope alleles. Conclusion. Despite a significant prevalence of anti-CCP in FDR, anti-PAD4 antibodies were almost exclusively found in established RA. The prevalence of anti-PAD4 antibodies in RA is similar to the prevalence described in other populations and these autoantibodies are associated with disease duration and anti-CCP in RA.

Aberrant Cellular Localization of Rubella Viral Genome in Patients with Adult Still's Disease-A Pilot Study

The rubella virus (RV) genome was detected using polymerase chain amplification techniques in several peripheral blood cell populations in patients with adult Stills disease (ASD) and normal controls (NC), including mononuclear cells (PBMC), B-cells, T-cells, monocyte/macrophages, and polymorphonuclear leukocytes (PMN). Five of 6 ASD patients and 3 of 6 NC subjects had detectable RV genome. Viral genomic load was significantly higher in ASD than in NC subjects (4.4 fold higher, p = 0.03). Interestingly, a differential cellular distribution of viral genome was observed between ASD and NC individuals. RV genome was detected more frequently in the PBMCs of ASD (5 of 6) patients compared to 2 of 6 NC. The viral genome was more localized to the PMN compartment equally in ASD and in NC subjects. On further cellular analysis, RV genome was detected in B-cells and macrophages but not T-cells in one patient. Existence of a differential viral genomic reservoir between ASD and NC suggests that this may play a role in the pathogenesis of disease manifestations and may reflect the inability to clear latent virus.