Epitope-specific anti-nuclear antibodies are expressed in a mouse model of primary biliary cirrhosis and are cytokine-dependent.

Although the hallmark of primary biliary cirrhosis (PBC) is the presence of anti-mitochondrial antibodies (AMA), a significant number of patients have anti-nuclear antibodies (ANA) directed primarily against two nuclear proteins, gp210 and sp100. In PBC, there are considerable data on the specificity of these anti-nuclear antibodies as well as suggestive evidence that antibodies to gp210 predict a poor outcome. However, a further understanding of the significance of these autoantibodies has been hampered by limitations in accessing human subjects in a preclinical or early asymptomatic stage. To overcome this limitation, we have taken advantage of transgenic mice with abrogated transforming growth factor-ß signalling in T cells (dnTGF-ßRII) that develop histological features of PBC as well as the same AMA specificity. We studied these mice for serum ANA, including specific autoantibodies against gp210 and sp100. We further examined sera from dnTGF-ßRII mice with concurrent deletions of the genes encoding interleukin (IL)-12p35, IL-12p40, IL-23p19, IL-17, IL-6, interferon (IFN)-γ or tumour necrosis factor (TNF)-α. Sera from all the dnTGF-ßRII mouse lines contained antibodies against gp210 and sp100. Of significance, mice with germline deletions of the genes encoding IL-12p40, IL-23p19, IL-17, IL-6 and TNF-α had significantly lower titres of anti-gp210 antibodies. These results provide a platform to dissect the mechanisms of gp210 and sp100 autoantibody production in dnTGF-ßRII mice as well as to study the possible role of ANA in the pathophysiology of PBC.

Immunoglobulin gene usage and immunohistochemical characteristics of human monoclonal antibodies to the mitochondrial autoantigens of primary biliary cirrhosis induced in the XenoMouse.

The immunodominant antimitochondrial antibody (AMA) response in primary biliary cirrhosis (PBC) is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). The nature of the clonal selection process is unclear, and to address this issue, we took advantage of a transgenic technology, XenoMouse, that contains 80% of the human immunoglobulin (Ig) variable gene repertoire and can produce high-affinity human antibodies to virtually any immunogen without evidence of clonal bias. We immunized mice with PDC-E2 to obtain 13 HmAbs, including 4 IgG(2) and 9 IgM isotypes. Immunoglobulin gene analysis was unique and demonstrated a clonal bias; the immunoglobulin gene usage was considerably different from other antibody responses analyzed in XenoMouse systems. Four of the 13 mAbs recognized the inner lipoyl domain of PDC-E2, 2 of 13 recognized the entire PDC-E2 molecule, 4 of 13 recognized PDC-E2 and OGDC-E2, 1 of 13 recognized OGDC only, 1 recognized BCOADC-E2 only, and 1 recognized an unidentified 100-kd mitochondrial protein. Immunohistochemical staining using these HmAbs produced mitochondrial staining of septal bile ducts in both PBC and control livers. Ig gene analysis showed that 7 of 13 HmAbs used the V(H)3 and 4 of 13 used VH4 gene repertoire, respectively. Three of 7 V(H)3 antibodies used the same Ig VH3-21 gene family found in human AMA from patients with PBC. The CDRs of these autoantibodies were slightly mutated when compared with the sequences present within the Ig germline genes. In conclusion, the XenoMouse not only recapitulates the unique specificity and restriction of PBC patients, but indicates that the autoantibodies are derived from a restricted clonal selection process. Such data suggest that the original immunogen leads to somatic mutation without subsequent development of determinant spreading.

Genomic analysis of differentially expressed genes in liver and biliary epithelial cells of patients with primary biliary cirrhosis.

The characterization of differentially expressed genes provides a powerful tool for identifying molecules that may be involved in the pathogenesis of disease. We have used two independent techniques to identify overexpressed transcripts in bile duct cells and in liver from patients with primary biliary cirrhosis (PBC). In the first method, we used suppressive subtractive hybridization to compare mRNA from isolated PBC bile duct epithelial cells (BECs) to normal BECs and identified 71 clones as transcribed at higher levels in PBC-BECs. Amongst these clones, 62/71 had matches in a non-redundant nucleotide database and 9/71 had matches in an EST database. Of the 62 clones, 51/62 include a complexity of genes involved in cell proliferation, signal transduction, transcription regulation, RNA processing, carbohydrate metabolism and hypothetical/unknown proteins; 4/62 were identified as interstitial collagenase and collagenase precursors, 4/62 as ribosomal proteins, 3/62 as mitochondrial DNA. The mitochondrial cDNA sequences included cytochrome c oxidase, Wnt-13, and the pHL gene, a c-myc oncogene containing coxIII sequence. In the second method, we constructed cDNA libraries from three different PBC livers and sequenced a total of 12,324 independent clones. These 12,324 clones underwent virtual subtraction with 2,814,148 independent clones from Incyte LifeSeq libraries. Twenty one sequences were identified as unique to PBC liver. Collectively, these approaches identified a number of genes involved in signalling, RNA processing, mitochondrial function, inflammation, and fibrosis. Interestingly, both Wnt-13 and Notch transcripts are overexpressed in PBC liver. Further studies are needed to focus on the significance of these genes during the natural history of disease.

Mutational analysis of immunoglobulin germline derived Vlambda4B light chains in rheumatoid arthritis.

OBJECTIVE: We investigated the somatic mutational pattern of a specific Vlambda light chain variable region (V) gene in rheumatoid arthritis (RA) patients. The Vlambda4B light chain was chosen because of its location on the lambda locus and because of its previously observed use in IgM rheumatoid factors. METHODS: We sequenced 13 different mRNA transcripts of Vlambda4B from the synovium of three different RA patients. These were compared to 31 identifiable Vlambda4B sequences from GenBank, which were obtained from the PBL of patients without RA. RESULTS: A subset of Vlambda4B had a high rate of mutation, especially in the framework regions within the RA synovium. Furthermore, a set of codons within the first complementary determining region of Vlambda4B displayed frequent replacement mutations but did not possess any silent mutations. CONCLUSION: The hypermutation of RA synovial-derived Vlambda4B sequences, especially in the framework areas, may contribute to or may be the result of altered mutational mechanisms and/or prolonged B cell life.

Mutational analysis of immunoglobulin germline derived Vlambda4A light chains in rheumatoid arthritis.

In order to better characterize the expression of a family of light chains previously expressed in IgM rheumatoid factors, we studied the usage and somatic mutational pattern of the Vlambda4A light chain gene in rheumatoid arthritis (RA) patients. We sequenced 11 different transcripts of Vlambda4A from the synovial tissue of three different RA patients. For comparison, we found 8 rearranged transcripts of Vlambda4A from 4 normal peripheral blood lymphocyte libraries and 1 rearranged transcript from a non-RA con-A-resistant hybridoma in GenBank. A previously undescribed polymorphism of Vlambda4A was noted. Furthermore, conserved replacement mutations in the complementary determining regions, common silent mutations around these replacement mutations, and two subsets of mutated sequences were detected in multiple RA patients. These mutation patterns also correlated with observed consistencies in the rearrangements of the Vlambda4A/Jlambda junction. These data suggest that there is clonal expansion of Vlambda4A light chains in the RA synovium in response to a RA-specific antigen or as the result of an idiotypic response in RA.

Analysis of the molecular basis of synovial rheumatoid factors in rheumatoid arthritis.

The objective of this study was to better understand the molecular basis of IgM rheumatoid factor in rheumatoid arthritis (RA). We recently generated 10 different monoclonal IgM RF (mRF) molecules isolated from the synovium of a single patient with RA. The heavy (H) and light chain (L) variable region (V) genes of these 10 mRFs were cloned and sequenced. Six mRFs used kappa light chains and 4 mRFs used lambda light chains. Of particular interest, 8 of 10 heavy chains used the JH4 joining region gene, and all five VH4 heavy chains used the DK4 diversity region gene with the JH4. Four of the VH4 clones used the same germline gene, likely representing a novel but closely related germline gene to VH4.18, and may be clonally related because of the extensive homology in their heavy chain sequence. Two VH4 clones shared the same light chain gene, VkappaIIIb kv325 (99% homology) and the same JK4 joining region gene, while three VH4 clones used two different light chain genes, an uncommon Vkappa4 and a Vlambda4 gene, respectively. In this RA patient, there was recurrent utilization of VH4-DK4-21/10-JH4 genes and a recurring association with gene elements Vkappa3 and Vlambda4. Recurring usage of Vkappa3 (kv325) and Vlambda4 (lv418) gene elements may result from a light chain editing process whereby immature autoreactive B cells encountering self-antigen attempt, and often succeed, in altering their specificities through secondary Ig light chain gene rearrangement. Moreover, the oligoclonality of these RFs suggest clonal relatedness secondary to an antigen-driven response.

Polymerase chain reaction and restriction endonuclease digestion for selected members of the "Mycoplasma mycoides cluster" and Mycoplasma putrefaciens.

A specific diagnostic method using the polymerase chain reaction, together with restriction endonuclease digestion patterns, was developed for members of the "Mycoplasma mycoides cluster" that normally occur in the United States (i.e., Mycoplasma mycoides subsp. mycoides Large Colony and Mycoplasma capricolum subsp. capricolum in addition to "cluster" mycoplasma, bovine serogroup 7, and Mycoplasma putrefaciens. The digestion of "cluster" polymerase chain reaction DNA (1,225 bp) amplification products with restriction enzymes AseI and SspI gave mycoplasma species-specific patterns for all strains of M. mycoides subsp. mycoides Large Colony, M. capricolum subsp. capricolum, and bovine group 7 tested. Moreover, we found a nonspecific amplification product for M. putrefaciens that occurred with the oligonucleotide primers used for the "M. mycoides cluster" reaction. However, the restriction endonuclease digestion patterns observed with the restriction enzymes AluI, AseI, and SspI for M. putrefaciens were different than the digestion patterns obtained for the other "cluster" mycoplasmas. This report confirms the usefulness of polymerase chain reaction DNA amplification allied with restriction enzyme digestion profile analysis for the rapid and specific identification of mycoplasmas belonging to the "M. mycoides cluster" and M. putrefaciens.

Immunologic mechanisms in common rheumatologic diseases.

Rheumatoid arthritis and seronegative spondyloarthropathies are rheumatologic diseases that likely are caused by inflammatory reactions occurring in genetically predisposed individuals mounting an immune response to the antigen. Understanding the immunopathology of these diseases provides insight into their etiology, pathogenesis, and a rationale for therapies targeting immune component interactions. Although the antigen in rheumatoid arthritis is not known, several bacterial antigens have been associated with seronegative spondyloarthropathies. These antigens result in an interaction between the human leukocyte antigen-B27 restricted CD8 positive T lymphocytes and the antigen presenting cell, producing an inflammatory response. Rheumatoid factors are autoantibodies directed against the fragment crystallizable portion of the immunoglobulin G. Rheumatoid factor immunoglobulin G immune complexes contribute to the inflammatory events in the rheumatoid joint, and may play an important role in antigen presentation. A novel antigen capture enzyme linked immunosorbent assay was developed that mimicked B cell surface expressed rheumatoid factor. Conversely, a direct binding enzyme linked immunosorbent assay mimicked secreted rheumatoid factor. Comparison of rheumatoid binding enzyme linked immunosorbent assays showed that the physical state of rheumatoid factor can affect binding characteristics. The state of glycosylation of immunoglobulin G may contribute to its antigenic structure. These physical characteristics may be important in rheumatoid factor's pathogenic role in rheumatoid arthritis.

Antigen binding differences between secreted and cell surface expressed rheumatoid factor derived from inflamed synovium.

OBJECTIVE: Rheumatoid factor (RF) is the predominant autoantibody in rheumatoid arthritis (RA). but its role in the pathogenesis of RA remains unclear. We hypothesized that surface RF (sRF) expressed on antigen presenting B cells (B-APC) might have different binding specificities than secreted RF. METHODS: We examined RF binding in a novel RF antigen capture enzyme linked immunoassay (ACE) that mimicked sRF binding, and compared it with a direct binding enzyme linked immunoassay (DBE) that mimicked secreted RF. RESULTS: Significant differences in binding characteristics by the same rheumatoid synovial cell (RSC) derived monoclonal RF (mRF) were observed between the ACE and the DBE. For example, several mRF that demonstrated the classical Ga binding pattern (binding to IgG1, 2, and 4) in the DBE showed considerable binding to selected IgG3 proteins in the ACE; and several mRF that bound only to rabbit IgG in DBE bound to human IgG in the ACE. CONCLUSION: These RF reactivity differences may be attributed to conformational modifications in the RF and IgG molecules that expose different epitopes or altered binding sites depending on the physical state of the antibody and/or antigen and may be important pathogenically.

Preferential utilization of a novel V lambda 3 gene in monoclonal rheumatoid factors derived from the synovial cells of rheumatoid arthritis patients.

OBJECTIVE: To further our understanding about the molecular genetics of rheumatoid factor (RF) in rheumatoid arthritis (RA). METHODS: The heavy and light chain variable region (V) genes of 5 new human monoclonal IgM RFs were cloned and sequenced using the polymerase chain reaction and the dideoxynucleotide termination method. RESULTS: The results reveal the recurrent usage in two RA patients of a novel V lambda 3 germline gene, designated Humlv3c93. Specifically, in 2 of 3 RFs (C93 and D53) from one patient, the light chains in the V lambda gene-encoded region were identical to each other and to the light chain of an RF (H4) from another patient. Serologically, the light chains of these 3 RFs were classified as members of the V lambda 3b sub-subgroup. Each of the RFs was encoded by a different VH gene. Both C93 and D53 bound specifically with human and rabbit IgG, whereas H4 was monospecific for rabbit IgG. CONCLUSION: Since the lv3c93 gene is not homologous to any reported V lambda sequence from natural autoantibodies, it is possible that lv3c93 may represent a disease-specific RF-related V lambda gene. Moreover, the amino acid sequence CSGGSCY in the third complementarity-determining regions of 2 of the RF heavy chains is encoded by the DLR2 gene segment and has been found previously in 2 other RA-derived RFs, and thus may play a significant role in antigen binding.

IgG3 reactive rheumatoid factor in rheumatoid arthritis: etiologic and pathogenic considerations.

Rheumatoid factor (RF) is a polyclonal autoantibody directed against the Fc portion of IgG. Although the role of RF in patients with rheumatoid arthritis (RA) is unclear, immune complexes that form between RF and IgG can activate the classical complement (C) pathway, leading to pathogenic outcomes involving inflammatory events and tissue damage. The specificity of serum RF and RF produced by rheumatoid synovial cells (RSC) is different. Serum RF has specificity for rabbit IgG and human IgG subclasses IgG1, 2, and 4, but binds poorly to IgG3. The affinity of serum RF for IgG Fc is low, having an association constant of 10(4)-10(5) M-1. RSC RF, however, has specificity for human IgG and high avidity for IgG3. Because of this greater specificity and avidity for IgG3, and because RSC RF may be pathogenically more important than serum RF, an important role for IgG3-reactive RF in RA may exist. Binding of RF to IgG may be dependent on the allotype and glycosylation of IgG. Infectious agents present in RA patients may directly or indirectly induce the production of certain RF. In this communication, we review and expand on several observations examining the role of IgG3-reactive RF in RA including: 1) binding differences between RF derived from RSC and serum; 2) glycosylation characteristics of IgG and its interaction with RF; 3) apparent allotype dependent binding of IgG3-reactive RF; and 4) possible relationship between infectious agents and the production of IgG3-reactive RF. Taken together, these observations suggest an important role for IgG3-reactive RF in better understanding the etiology and pathogenesis of RA.

Allotypic dependency of the specificity and avidity of human monoclonal IgM rheumatoid factors derived from rheumatoid synovial cells.

OBJECTIVE: To better understand the genetic derivation and pathogenicity of rheumatoid factor (RF) molecules in rheumatoid arthritis (RA), we have focused our studies on rheumatoid synovial cells (RSC). METHODS: Five monoclonal human IgM rheumatoid factor (mRF)-secreting hybridomas were produced from the RSC of an RA patient. Fine subclass specificities and avidities of these RSC mRFs were compared with several paraprotein monoclonal IgM RFs using direct binding (reactivity) and competitive inhibition (specificity and avidity) enzyme-linked immunosorbent assays. RESULTS: The following observations were made: 1) RSC mRF had greater avidity for IgG than did paraprotein mRF; 2) 4 of the 5 RSC RF were highly avid for IgG3; and, 3) the avidity of RSC RF binding for IgG3 was highest for IgG molecules expressing the G3m(5) allotype. CONCLUSION: We conclude that RSC RF have different specificities and avidities than do paraprotein RF. This may suggest an antigen-driven process in RA synovium, with the production of higher-avidity IgG3m(5)-specific RSC RF, which could have special pathogenetic importance.

Molecular analysis of rheumatoid factors derived from rheumatoid synovium suggests an antigen-driven response in inflamed joints.

OBJECTIVE: Understanding the molecular genetic basis for rheumatoid factor (RF) production is necessary to a better understanding of the etiology and pathogenesis of rheumatoid arthritis (RA). We sought to define the genetic basis of RF in RA. METHODS: The heavy and light chain variable region genes encoding 4 human monoclonal RF were cloned and sequenced using the polymerase chain reaction and the dideoxynucleotide chain-termination method. RESULTS: The heavy and light chains of the C6 RF and the light chain of the G9 RF were encoded by 3 new RF-related Ig V-region genes. The heavy and light chains of D5 and G4 RFs were identical; most of their mutations caused amino acid substitutions. CONCLUSIONS: The RF-related Ig V-region gene repertoire is large and is still expanding. The data from D5 and G4 strongly suggest that these 2 RFs arise in an antigen-driven response in rheumatoid synovium. The presumed germline V genes for C6 may represent disease-specific RF-related V genes.

A systematic approach to defining the germline gene counterparts of a mutated autoantibody from a patient with rheumatoid arthritis.

OBJECTIVE: To define the germline counterparts of potentially highly mutated autoantibodies in disease states. METHODS: We developed a systematic approach by first characterizing a rearranged Ig gene and its upstream flank, and then designing suitable primers to amplify specifically the putative germline counterpart. RESULTS: We identified and characterized the germline counterpart of a rheumatoid factor heavy chain variable region. CONCLUSION: We showed unequivocally that the heavy chain of a rheumatoid factor, derived from synovial tissue, has 4 replacement mutations from the corresponding germline gene. The technique allows quick assessment of the degree of somatic mutation in many autoantibodies, and thus can help to elucidate the underlying mechanisms for the induction and sustained production of such antibodies in patients.

Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains.

Desmosomal glycoproteins 2 and 3 (dg2 and 3) or desmocollins have been implicated in desmosome adhesion. We have obtained a 5.0-kb-long clone for dg3 from a bovine nasal epidermal lambda gt11 cDNA library. Sequence analysis of this clone reveals an open reading frame of 2,517 bases encoding a polypeptide of 839 amino acids. The sequence consists of a signal peptide of 28 amino acids, a precursor sequence of 104 amino acids, and a mature protein of 707 amino acids. The latter has the characteristics of a transmembrane glycoprotein with an extracellular domain of 550 amino acids and a cytoplasmic domain of 122 amino acids. The sequence of a partial clone from the same library shows that dg2 has an alternative COOH terminus that is extended by 54 amino acids. Genomic DNA sequence data show that this arises by splicing out of a 46-bp exon that encodes the COOH-terminal 11 amino acids of dg3 and contains an in-frame stop codon. The extracellular domain of dg3 shows 39.4% protein sequence identity with bovine N-cadherin and 28.4% identity with the other major desmosomal glycoprotein, dg1, or desmoglein. The cytoplasmic domain of dg3 and the partial cytoplasmic domain of dg2 show 23 and 24% identity with bovine N-cadherin, respectively. The results support our previous model for the transmembrane organization of dg2 and 3 (Parrish, E.P., J.E. Marston, D.L. Mattey, H.R. Measures, R. Venning, and D.R. Garrod. 1990. J. Cell Sci. 96:239-248; Holton, J.L., T.P. Kenny, P.K. Legan, J.E. Collins, J.N. Keen, R. Sharma, and D.R. Garrod. 1990. J. Cell Sci. 97:239-246). They suggest that these glycoproteins are specialized for calcium-dependent adhesion in their extracellular domains and, cytoplasmically, for the molecular interactions involved in desmosome plaque formation. Moreover this represents the first example of alternative splicing within the cadherin family of cell adhesion molecules.

Desmosomal glycoproteins 2 and 3 (desmocollins) show N-terminal similarity to calcium-dependent cell-cell adhesion molecules.

The N-terminal sequence of a mixture of desmosomal glycoproteins 2 and 3 (dg2/3, desmocollins) from bovine nasal epidermis, prepared by electro-elution from polyacrylamide gels, was determined by solid-phase Edman degradation. A sequence of 23 amino acids was obtained. This showed 43% identity with that of the N terminus of the calcium-dependent cell adhesion molecule, N-cadherin. A lesser degree of identity with other members of the cadherin-uvomorulin-L-CAM family was also found. In order to confirm that the sequence was derived from the dg2/3 molecules a rabbit antiserum was raised against a synthetic peptide corresponding to the sequence, conjugated to keyhole limpet haemocyanin (KLH). The antiserum obtained showed high (titre) activity against both the peptide and KLH in ELISA. Each activity could be specifically adsorbed with the appropriate ligand. The antiserum reacted specifically with both dg2 and dg3 of bovine nasal epidermis on immunoblots, this binding was blocked by the N-terminal peptide but was unaffected by KLH. The identity of dg2 and -3 in these preparations was confirmed by immunoblotting with two monoclonal antibodies and one polyclonal antiserum raised against the whole molecules. The N-terminal peptide antiserum was shown to bind to the intercellular space of desmosome profiles by immunoelectron microscopy on ultra-thin frozen sections. One of the two monoclonal antibodies (07-4D) also reacted with the desmosomal intercellular space. dg2 and -3 were shown by Staphylococcus aureus V8 protease digestion to have identical one-dimensional peptide maps. Both the N-terminal antiserum and 07-4D reacted with a V8 fragment of 19,000 Mr derived from dg2 and dg3.(ABSTRACT TRUNCATED AT 250 WORDS)

Serologic and molecular characterization of a human monoclonal rheumatoid factor derived from rheumatoid synovial cells.

Molecular characterization of rheumatoid factors (RF) in rheumatoid arthritis (RA) has been hampered because of their polyclonality. To overcome this problem, we generated monoclonal RF-secreting hybridomas from rheumatoid synovial cells. Among the RF-secreting hybridomas, HAF10 secreted an IgM-RF that was monospecific for human IgG. It bound well to IgG1 and IgG2, but not to IgG3 and IgG4. Sequence analysis of its heavy and light chains showed that it contained a VH1 heavy chain and a V lambda light chain that did not belong to any known lambda light chain subgroup, and therefore, probably represented a new lambda subgroup. These results indicated that both the heavy and light chains of a monoclonal IgM-RF from rheumatoid synovial cells were quite different from the reported variable region sequences of several monoclonal RF derived mainly from patients with mixed cryoglobulinemia. Further studies of additional monoclonal RF from RA patients are warranted to define precisely their genetic basis and to further our understanding of the immunopathology of RA.