International Archives of Allergy and Immunology, 1983
Mast cells can be dispersed from human tonsils by a mechanical procedure or by the use of proteol... more Mast cells can be dispersed from human tonsils by a mechanical procedure or by the use of proteolytic enzymes. The latter procedure does not always increase cell yield and may adversely affect the mast cell response to secretory stimuli. Mechanically dispersed tonsillar mast cells secrete histamine in response to stimulation with anti-IgE and calcium ionophore A23187 by a calcium-dependent and energy-dependent mechanism. With compound 48/80 and basic polyamines, histamine release only occurs at cytotoxic concentrations. Functionally, human tonsillar mast cells are similar to adenoidal and lung mast cells.
In previous studies we have demonstrated high levels of rheumatoid factor (RF) and large-size (gr... more In previous studies we have demonstrated high levels of rheumatoid factor (RF) and large-size (greater than 22S) circulating immune complexes (CIC) in the serum of rheumatoid arthritis (RA) patients with extra-articular disease. These findings were paralleled by a concurrent increase in the level of RF-associated cross-reactive idiotypes (CRI) and an apparent diversification of the RF repertoire detected in the serum of the same patients. In the present study we examine the ability of CICs to activate the complement system in vivo, and its possible influence on expanding the RF repertoire in RA patients with extra-articular disease. Activation of complement by CICs is the key for germinal centre localization and long-term retention of such complexes on the surface of follicular dendritic cells (FDC), and so provides a source for the selection of cells with high affinity receptors for IgG and leads to the establishment of immunological memory. CICs containing different immunoglobulin isotypes and from different patients localized in mouse spleen germinal centres. However, intense localization was mainly seen for IgG-containing complexes from the serum of patients with large-size (greater than 22S) IgG-IgM RF complexes. The ability of these complexes to localize in mouse spleen germinal centres was related to activation of the complement system via the classical pathway in the patients' sera. Localization of IgG complexes was significantly (P less than 0.05) higher in sera from RA patients with extra-articular disease than those with articular disease alone. This study demonstrates the ability of large-size (greater than 22S) IgG-IgM RF complexes to activate complement, and suggests a possible role for such complexes in modulating the immune response to IgG in RA patients with extra-articular disease.
7B4, B6 and D12 are murine monoclonal antibodies (MoAb) that bind to some human immunoglobulin he... more 7B4, B6 and D12 are murine monoclonal antibodies (MoAb) that bind to some human immunoglobulin heavy chain products of the closely related V3–30, V3–30.3 and V3–33 genes from the VH3 family. B6 and D12 have additional reactivities with some immunoglobulins (Ig) encoded by the V3–11 and V3–7 genes; D12 also reacts with some V3–43 gene Ig. We show here, by site‐directed mutagensis, that the lysine at position 57 in the complementarity‐determining region 2 (CDR‐2) of the V3–30 gene product is crucial for epitope recognition by all three anti‐VH3 MoAbs. Further analysis of the amino‐acid sequences of a large panel of Ig reactive, or nonreactive, with MoAb 7B4 indicates that the determinant recognized by 7B4 is dependent on the presence of the tetrapeptide sequence NKYY between positions 56 and 59 in the CDR‐2. Comparing the efficiency of 7B4 reactivity with VH3 gene‐encoded human Ig indicates that amino‐acid position 4 in the frame region 1 (FR‐1) may also influence the binding of 7B4 to Ig encoded by three very closely related germline genes, V3–30, V3–30.3 and V3–33. NKYY is also found on the gp120 V3 region of human immunodeficiency virus (HIV)‐2, SIV and HTLV‐4. We also report that other tetrapeptide sequences found on the 56–59 motif of heavy chain variable regions encoded by germline genes are expressed on the solvent exposed V2 region of gp120 of HIV‐1 isolates. The possible significance of these observations is discussed.
To elucidate the molecular basis for the ability of antibodies encoded by the human V~2 6 heavych... more To elucidate the molecular basis for the ability of antibodies encoded by the human V~2 6 heavychain variable region gene to react with diverse antigens, we have generated 34 hybridomas secreting chimaeric monoclonal antibodies (human p heavy chain/mouse light chains) from transgenic mice. The transgenic mice carry an immunoglobulin minilocus containing the human V~2 6 gene, human DH and JH gene segments, and genes encoding the human Cp region. The minilocus in these animals undergoes functional rearrangement resulting in the production of chimaeric antibodies in which human p heavy chains utilizing the VH26 gene are paired with mouse K or 1 light chains. The hybridomas described in this study were generated from naTve animals and were selected solely on the basis of human p-chain expression. The antibodies described have covalently attached mouse light chains and are multimeric in structure. The binding properties of the antibodies were examined using a panel of both self-and foreign antigens using enzyme-linked immunosorbent assays, agglutination or radio-immunoprecipitation assays and immunofluorescence. Chimaeric-immunoglobulins from 21 of the 34 hybridoma clones (61.7%) reacted with one or more antigens, of which 13 (38.2%) reacted with more than two antigens. These studies demonstrate that the V~2 6 gene, in combination with human DH and JH gene segments, and mouse light-chain genes, is able to encode antibodies with a wide range of ligand-binding specificities. These findings have important implications in the context of the possible origins of autoantibodies encoded by V~2 6 which may play a role in the pathogenesis of a number of autoimmune conditions.
Sixteen lymph nodes from 14 patients with rheumatoid arthritis were examined immunohistochemicall... more Sixteen lymph nodes from 14 patients with rheumatoid arthritis were examined immunohistochemically and morphometrically and compared with 10 control nodes showing follicular hyperplasia from patients without rheumatoid disease. Frozen material was available from nine patients and all controls. The nodes from patients with rheumatoid arthritis seemed to share characteristic features. The most striking of these was follicular hyperplasia in which the germinal centres, in spite of being quite large, showed relatively sparse proliferative activity. The nodes often showed infiltration of germinal centres by CD8 positive T lymphocytes and contained fewer IL2R positive cells in the paracortex than controls. These and other features may have some correlation with disease activity. Lymphadenopathy in rheumatoid arthritis may not just be a manifestation of joint inflammation but an active component of this multisystem disease and may reflect a widespread immunological abnormality.
The production of hybridomas derived from CD5+ B lymphocytes is important for studying the immuno... more The production of hybridomas derived from CD5+ B lymphocytes is important for studying the immunoglobulin gene repertoire and antibody specificity of this B-lymphocyte subpopulation. However, hybridomas derived from these lymphocytes invariably lose surface membrane expression of CD5 following hybridization. This impedes the unequivocal assignment of the generated hybridomas to the CD5+ B-lymphocyte subpopulation from unsorted, or partially sorted, cells. Recent studies have shown that mRNA transcripts of the CD5 gene and the protein product can be detected in the cytoplasm of some mouse hybridomas, indicating that although surface expression has been lost, they may have been generated from CD5+ B lymphocytes. These studies, however, have been unable to discount completely the possibility that aberrant cytoplasmic expression of the CD5 gene occurred as a direct consequence of the hybridization process. To confirm that cytoplasmic CD5 expression in the hybridomas is in fact directly related to the B-lymphocyte origin we have generated hybridomas from FACS-sorted CD5+ and CD5- murine splenic B lymphocytes, and determined their surface and cytoplasmic CD5 expression by fluorescence-activated cell sorting. Our findings reveal that all hybridomas derived from CD5+ B lymphocytes (nine hybridomas) were negative for surface but positive for cytoplasmic CD5 expression, whereas all hybridomas derived from CD5- B lymphocytes (nine hybridomas) were negative for both surface and cytoplasmic CD5 expression. This finding shows that staining for cytoplasmic CD5 expression provides an accurate method of determining the cellular origin of murine B-lymphocyte hybridomas.
T cell hybridomas HCQ6 and MD.45 acquired Ab-type specificity to collagen type II, when engrafted... more T cell hybridomas HCQ6 and MD.45 acquired Ab-type specificity to collagen type II, when engrafted with a chimeric cell surface receptor, scC2Fv/gamma, which includes the single-chain Fv domain (scFv) of the anti-collagen type II mAb C2 and the signaling gamma subunit of the Fc epsilonRI. When transduced into MD.45 cells, scC2Fv/gamma or its mutated form lacking immunoreceptor tyrosine-based activation motif (ITAM), scC2Fv/gammaIC-, formed mainly homodimers. A small proportion of these molecules formed heterodimers with endogenous CD3zeta in these hybridoma cells. By contrast, in HCQ6 cells, the majority of scC2Fv/gamma and scC2Fv/gammaIC- molecules formed heterodimers with CD3zeta, and only a small proportion of them was expressed as homodimers. Stimulation with plastic-immobilized collagen induced IL-2 production in scC2Fv/gamma-transduced MD.45 cells, but not in MD.45 cells transduced with the ITAM-less chimera scC2Fv/gammaIC-. HCQ6 cells transduced with scC2Fv/gamma responded to plastic-bound collagen. Due to the high content of CD3zeta-associated chimeras, HCQ6 cells transduced with the ITAM-less scC2Fv/gammaIC- chimera were also responsive to plastic-bound collagen. When cells were stimulated with collagen in solution, MD.45 cells transduced with scC2Fv/gamma produced IL-2, whereas transduced HCQ6 cells were unresponsive, hence suggesting that the ability of cells transduced with scC2Fv chimeras to respond to soluble collagen correlated with predominant expression of divalent scC2Fv/gamma homodimers, but not monovalent scC2Fv/gamma-CD3zeta or scC2Fv/gammaIC(-)-CD3zeta heterodimers. Of interest, expression of CD3 subunits in hybridomas transduced with scC2Fv chimeras was reduced, resulting in decreased response to cognate Ags.
Systemic sclerosis (SSc) is a complex, immune-mediated rheumatic disease characterised by excessi... more Systemic sclerosis (SSc) is a complex, immune-mediated rheumatic disease characterised by excessive extracellular matrix deposition in the skin and internal organs. B cell infiltration into lesional sites such as the alveolar interstitium and small blood vessels, alongside the production of defined clinically relevant autoantibodies indicates that B cells play a fundamental role in the pathogenesis and development of SSc. This is supported by B cell and fibroblast coculture experiments revealing that B cells directly enhance collagen and extracellular matrix synthesis in fibroblasts. In addition, B cells from SSc patients produce large amounts of profibrotic cytokines such as IL-6 and TGF-b, which interact with other immune and endothelial cells, promoting the profibrotic loop. Furthermore, total B cell counts are increased in SSc patients compared with healthy donors and specific differences can be found in the content of naïve, memory, transitional and regulatory B cell compartments. B cells from SSc patients also show differential expression of activation markers such as CD19 which may shape interactions with other immune mediators such as T follicular helper cells and dendritic cells. The key role of B cells in SSc is further supported by the therapeutic benefit of B cell depletion with rituximab in some patients. It is notable also that B cell signaling is impaired in SSc patients, and this could underpin the failure to induce tolerance in B cells as has been shown in murine models of scleroderma.
consecutive patients attending the outpatient and in-patient departments of the Department of Rhe... more consecutive patients attending the outpatient and in-patient departments of the Department of Rheumatology & Clinical Immunology at our institute, classified as SSc as per the ACR/EULAR 2013 criteria, were included after taking their informed consent. Age and gendermatched healthy controls were included and all enrolled subjects were subjected to a dual energy X-ray absorptiometry (DEXA) scan. Demographic and clinical details of all patients were recorded. Osteoporosis was defined as per the WHO definition of a T score-2.5. The study was approved by the Institutional Review Board and the Institutional Ethics Committee. Results 90 patients with SSc and 45 age and gender-matched healthy controls were included in the study. Confounding factors for bone health, like smoking or alcohol intake, the proportion of post-menopausal women, and past or family history of fragility fractures, were matched between the two groups. The prevalence of osteoporosis among SSc cases was significantly higher than in the healthy controls (34.4% vs. 6.6%, p < 0.001). In univariate analysis, age, low body mass index (BMI), post-menopausal status, diffuse cutaneous phenotype, gastrointestinal involvement, digital ulcers, and pulmonary arterial hypertension were found to be significantly associated with osteoporosis. Multivariate analysis revealed that the presence of osteoporosis was significantly associated with lower BMI, post-menopausal status, and the presence of digital ulcers in patients. Interestingly, glucocorticoid use did not appear to increase the risk of osteoporosis in our cohort. Conclusion Patients with systemic sclerosis and digital ulcers, low BMI, or postmenopausal status are at high risk of developing osteoporosis and therefore should be screened for osteoporosis by DEXA scan to prevent unforeseen fracture-related morbidity and mortality. Glucocorticoids may not be the major culprit as evidenced by this study, while control of disease activity may lead to better bone health status.
To define mechanisms of sustained activation of synovial B lymphocytes in RA, we studied hybridom... more To define mechanisms of sustained activation of synovial B lymphocytes in RA, we studied hybridomas established from the local synovial B cell repertoire of two RA patients for Vκ gene expression and for antigen-binding specificity. The analyses revealed that members of the main Vκ families (I, II and III) were utilized at frequencies consistent with random Vκ gene family use. Furthermore, although the hybridomas expressed genes frequently seen in response to other self- and exogenous antigens, only one VκI- and two of three VκIII-expressing hybridomas exhibited reactivity with self-antigens. Nucleotide sequence analysis revealed that all hybridomas, with the exception of rheumatoid factor (RF)-producing hybridomas, expressed Vκ genes highly related to known germ-line genes (99.3–100% homology) and that diversity was generated by deletions and random nucleotide insertions at the Vκ–Jκ junction. Examination of the few nucleotide changes seen within the Vκ genes revealed a predominance of silent to replacement changes. Moreover, most of these changes can be attributable either to allotypic variations or to limited random nucleotide replacements independent of antigen selection. In contrast, one IgG-RF (B4D8) exhibited predominantly replacement nucleotide changes in the complementarity-determining regions, suggestive of antigen-driven selection. The random expression of immunoglobulin variable region genes with no, or little, evidence of mutation in the synovial B lymphocyte repertoire, including natural polyreactive antibodies, alongside mutated IgG-RF, suggest that both polyclonal activation and antigen-driven responses occur in RA synovia.
The presence of germinal centre-like structures and clonotypic expansion of lymphocytes in RA syn... more The presence of germinal centre-like structures and clonotypic expansion of lymphocytes in RA synovia may indicate a site-specific immune response to local antigens, rather than passively entrapped immune cells, that sustains synovial inflammation. In this study we compare the nature of immunoglobulin light chain variable region gene use in the synovium of RA patients with peripheral B cells to determine the nature of the synovial immune response. Using Vl and Vk gene fingerprinting, which relies on differences in CDR3 length, we demonstrate differences in the pattern of Vl and Vk use and clonotypic expansion of B cells between the synovium and peripheral blood of RA patients. Further, we show that some synovial rearrangements with long CDR3 are selectively expanded. These longer than usual CDR3 were generated by a number of mechanisms including N-additions. However, the observed differences were not uniform in different patients. These observations suggest that local synovial antigens drive significant numbers of T and B lymphocytes selected from an existing repertoire shaped by genetic and environmental factors. Further, the data argue against passive retention of most B cells in the synovium of RA patients.
The NZB mouse is genetically predisposed to develop, at approximately 6 months of age, a spontane... more The NZB mouse is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti‐mouse erythrocyte auto‐antibodies. Molecular analysis of a panel of five anti‐erythrocyte monoclonal antibodies (MoAb) derived from splenocytes of unimmunized NZB mice revealed that these autoantibodies all had functionally rearranged genes from the VH J558 family of immunoglobulin genes with closest homology to germline genes H10 and H30. Owing to clustering of nucleotide differences within the CDRs, compared with the germline, it was concluded that these antibodies were most likely generated by an antigen‐driven mechanism.We report here further molecular analysis of two (4.16.1 and B4.13.2) of the panel of five anti‐mouse erythrocyte producing hybridomas which are apparently clonally related. Nucleotide analysis of the light chain cDNA indicated that both antibodies had closest homology to germline gene Vk24 and use Jk2 gene. Determination of the functional affinities of the MoAb reveal that B4.13.2 (IgG2a) has a &gt; 10‐fold higher affinity for mouse erythrocytes when compared to 4.16.1 (IgGl). This finding supports the view that these two autoantibodies are generated by an antigen‐driven mechanism. The proposed mechanism would involve the selection and expansion of a small population of B‐lymphocytes by antigen leading to isotype switch, somatic mutation and increased affinity. Our data also point to the possibility that some framework residues may be involved in the binding to antigen.
The New Zealand black (NZB) mouse strain is genetically predisposed to develop, at approximately ... more The New Zealand black (NZB) mouse strain is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti-mouse erythrocyte autoantibodies. In order to investigate the molecular mechanisms which lead to anti-mouse erythrocyte autoantibody production we have generated eight anti-mouse erythrocyte MoAbs producing hybridomas from splenocytes of 9-and 12-month-old NZB with spontaneous autoimmune anaemia. IgG2a was the predominant isotype, while IgM, IgGl and IgG2b were each produced by one hybridoma cell line. All anti-mouse erythrocyte MoAbs were characterized for their antigen specificities. None of the MoAbs cross-reacted with ss-or dsDNA or with other species' erythrocytes, with the exception of one MoAb which cross-reacted with rat erythrocytes. None ofthe eight hybridomas was demonstrated to express surface or cytoplasmic CD5, suggesting that they derived from CD5" B lymphocytes. All hybridomas when implanted intraperitoneally into BALB/c mice caused anaemia. In order to define the genetic basis and investigate the molecular mechanisms resulting in pathogenic anti-mouse erythrocyte autoantibody production, the pattern of immunoglobulin variable region gene use has been studied. Five ofthe eight MoAbs whose IgVH genes were sequenced all have functionally rearranged genes from the VH J558 gene family. There is evidence for somatic point mutations in the complementarity-determining regions (CDR) ofthe IgVH genes in all of these five MoAbs when compared with the closest known germline gene. We suggest that these nucleotide sequence changes are likely to reflect selection by an antigen-driven mechanism. Furthermore, the data indicate that pathogenic anti-mouse erythrocytes are not derived from 'natural' autoantibodies.
The specificities of a panel of erythrocyte-reactive MoAbs derived from NZB mice with autoimmune ... more The specificities of a panel of erythrocyte-reactive MoAbs derived from NZB mice with autoimmune haemolytic anaemia (AIHA) were determined by immunoprecipitation and immunoblotting. Of the eight antibodies, two (IgG1 MoAb 105-2H and IgG2a MoAb 34-3C) immunoprecipitated a 105-kD component identified as the erythrocyte anion channel band 3. A similar band was also immunoprecipitated by the IgG2b MoAb 34-2B when used at relatively high concentrations, but none of the remaining hybridoma antibodies precipitated any labelled erythrocyte components. In immunoblotting experiments only 34-2B reacted with band 3, indicating that the epitope recognized by this MoAb is robust and differs from the determinant(s) recognized by 105-2H and 34-3C. The remaining MoAbs to react by immunoblotting were the IgM antibodies IE10 and 4C8, both of which bound to a doublet corresponding to band 4.1 from the internal erythrocyte membrane skeleton. Of the three MoAbs which gave negative results in immunoprecip...
International Archives of Allergy and Immunology, 1983
Mast cells can be dispersed from human tonsils by a mechanical procedure or by the use of proteol... more Mast cells can be dispersed from human tonsils by a mechanical procedure or by the use of proteolytic enzymes. The latter procedure does not always increase cell yield and may adversely affect the mast cell response to secretory stimuli. Mechanically dispersed tonsillar mast cells secrete histamine in response to stimulation with anti-IgE and calcium ionophore A23187 by a calcium-dependent and energy-dependent mechanism. With compound 48/80 and basic polyamines, histamine release only occurs at cytotoxic concentrations. Functionally, human tonsillar mast cells are similar to adenoidal and lung mast cells.
In previous studies we have demonstrated high levels of rheumatoid factor (RF) and large-size (gr... more In previous studies we have demonstrated high levels of rheumatoid factor (RF) and large-size (greater than 22S) circulating immune complexes (CIC) in the serum of rheumatoid arthritis (RA) patients with extra-articular disease. These findings were paralleled by a concurrent increase in the level of RF-associated cross-reactive idiotypes (CRI) and an apparent diversification of the RF repertoire detected in the serum of the same patients. In the present study we examine the ability of CICs to activate the complement system in vivo, and its possible influence on expanding the RF repertoire in RA patients with extra-articular disease. Activation of complement by CICs is the key for germinal centre localization and long-term retention of such complexes on the surface of follicular dendritic cells (FDC), and so provides a source for the selection of cells with high affinity receptors for IgG and leads to the establishment of immunological memory. CICs containing different immunoglobulin isotypes and from different patients localized in mouse spleen germinal centres. However, intense localization was mainly seen for IgG-containing complexes from the serum of patients with large-size (greater than 22S) IgG-IgM RF complexes. The ability of these complexes to localize in mouse spleen germinal centres was related to activation of the complement system via the classical pathway in the patients&#39; sera. Localization of IgG complexes was significantly (P less than 0.05) higher in sera from RA patients with extra-articular disease than those with articular disease alone. This study demonstrates the ability of large-size (greater than 22S) IgG-IgM RF complexes to activate complement, and suggests a possible role for such complexes in modulating the immune response to IgG in RA patients with extra-articular disease.
7B4, B6 and D12 are murine monoclonal antibodies (MoAb) that bind to some human immunoglobulin he... more 7B4, B6 and D12 are murine monoclonal antibodies (MoAb) that bind to some human immunoglobulin heavy chain products of the closely related V3–30, V3–30.3 and V3–33 genes from the VH3 family. B6 and D12 have additional reactivities with some immunoglobulins (Ig) encoded by the V3–11 and V3–7 genes; D12 also reacts with some V3–43 gene Ig. We show here, by site‐directed mutagensis, that the lysine at position 57 in the complementarity‐determining region 2 (CDR‐2) of the V3–30 gene product is crucial for epitope recognition by all three anti‐VH3 MoAbs. Further analysis of the amino‐acid sequences of a large panel of Ig reactive, or nonreactive, with MoAb 7B4 indicates that the determinant recognized by 7B4 is dependent on the presence of the tetrapeptide sequence NKYY between positions 56 and 59 in the CDR‐2. Comparing the efficiency of 7B4 reactivity with VH3 gene‐encoded human Ig indicates that amino‐acid position 4 in the frame region 1 (FR‐1) may also influence the binding of 7B4 to Ig encoded by three very closely related germline genes, V3–30, V3–30.3 and V3–33. NKYY is also found on the gp120 V3 region of human immunodeficiency virus (HIV)‐2, SIV and HTLV‐4. We also report that other tetrapeptide sequences found on the 56–59 motif of heavy chain variable regions encoded by germline genes are expressed on the solvent exposed V2 region of gp120 of HIV‐1 isolates. The possible significance of these observations is discussed.
To elucidate the molecular basis for the ability of antibodies encoded by the human V~2 6 heavych... more To elucidate the molecular basis for the ability of antibodies encoded by the human V~2 6 heavychain variable region gene to react with diverse antigens, we have generated 34 hybridomas secreting chimaeric monoclonal antibodies (human p heavy chain/mouse light chains) from transgenic mice. The transgenic mice carry an immunoglobulin minilocus containing the human V~2 6 gene, human DH and JH gene segments, and genes encoding the human Cp region. The minilocus in these animals undergoes functional rearrangement resulting in the production of chimaeric antibodies in which human p heavy chains utilizing the VH26 gene are paired with mouse K or 1 light chains. The hybridomas described in this study were generated from naTve animals and were selected solely on the basis of human p-chain expression. The antibodies described have covalently attached mouse light chains and are multimeric in structure. The binding properties of the antibodies were examined using a panel of both self-and foreign antigens using enzyme-linked immunosorbent assays, agglutination or radio-immunoprecipitation assays and immunofluorescence. Chimaeric-immunoglobulins from 21 of the 34 hybridoma clones (61.7%) reacted with one or more antigens, of which 13 (38.2%) reacted with more than two antigens. These studies demonstrate that the V~2 6 gene, in combination with human DH and JH gene segments, and mouse light-chain genes, is able to encode antibodies with a wide range of ligand-binding specificities. These findings have important implications in the context of the possible origins of autoantibodies encoded by V~2 6 which may play a role in the pathogenesis of a number of autoimmune conditions.
Sixteen lymph nodes from 14 patients with rheumatoid arthritis were examined immunohistochemicall... more Sixteen lymph nodes from 14 patients with rheumatoid arthritis were examined immunohistochemically and morphometrically and compared with 10 control nodes showing follicular hyperplasia from patients without rheumatoid disease. Frozen material was available from nine patients and all controls. The nodes from patients with rheumatoid arthritis seemed to share characteristic features. The most striking of these was follicular hyperplasia in which the germinal centres, in spite of being quite large, showed relatively sparse proliferative activity. The nodes often showed infiltration of germinal centres by CD8 positive T lymphocytes and contained fewer IL2R positive cells in the paracortex than controls. These and other features may have some correlation with disease activity. Lymphadenopathy in rheumatoid arthritis may not just be a manifestation of joint inflammation but an active component of this multisystem disease and may reflect a widespread immunological abnormality.
The production of hybridomas derived from CD5+ B lymphocytes is important for studying the immuno... more The production of hybridomas derived from CD5+ B lymphocytes is important for studying the immunoglobulin gene repertoire and antibody specificity of this B-lymphocyte subpopulation. However, hybridomas derived from these lymphocytes invariably lose surface membrane expression of CD5 following hybridization. This impedes the unequivocal assignment of the generated hybridomas to the CD5+ B-lymphocyte subpopulation from unsorted, or partially sorted, cells. Recent studies have shown that mRNA transcripts of the CD5 gene and the protein product can be detected in the cytoplasm of some mouse hybridomas, indicating that although surface expression has been lost, they may have been generated from CD5+ B lymphocytes. These studies, however, have been unable to discount completely the possibility that aberrant cytoplasmic expression of the CD5 gene occurred as a direct consequence of the hybridization process. To confirm that cytoplasmic CD5 expression in the hybridomas is in fact directly related to the B-lymphocyte origin we have generated hybridomas from FACS-sorted CD5+ and CD5- murine splenic B lymphocytes, and determined their surface and cytoplasmic CD5 expression by fluorescence-activated cell sorting. Our findings reveal that all hybridomas derived from CD5+ B lymphocytes (nine hybridomas) were negative for surface but positive for cytoplasmic CD5 expression, whereas all hybridomas derived from CD5- B lymphocytes (nine hybridomas) were negative for both surface and cytoplasmic CD5 expression. This finding shows that staining for cytoplasmic CD5 expression provides an accurate method of determining the cellular origin of murine B-lymphocyte hybridomas.
T cell hybridomas HCQ6 and MD.45 acquired Ab-type specificity to collagen type II, when engrafted... more T cell hybridomas HCQ6 and MD.45 acquired Ab-type specificity to collagen type II, when engrafted with a chimeric cell surface receptor, scC2Fv/gamma, which includes the single-chain Fv domain (scFv) of the anti-collagen type II mAb C2 and the signaling gamma subunit of the Fc epsilonRI. When transduced into MD.45 cells, scC2Fv/gamma or its mutated form lacking immunoreceptor tyrosine-based activation motif (ITAM), scC2Fv/gammaIC-, formed mainly homodimers. A small proportion of these molecules formed heterodimers with endogenous CD3zeta in these hybridoma cells. By contrast, in HCQ6 cells, the majority of scC2Fv/gamma and scC2Fv/gammaIC- molecules formed heterodimers with CD3zeta, and only a small proportion of them was expressed as homodimers. Stimulation with plastic-immobilized collagen induced IL-2 production in scC2Fv/gamma-transduced MD.45 cells, but not in MD.45 cells transduced with the ITAM-less chimera scC2Fv/gammaIC-. HCQ6 cells transduced with scC2Fv/gamma responded to plastic-bound collagen. Due to the high content of CD3zeta-associated chimeras, HCQ6 cells transduced with the ITAM-less scC2Fv/gammaIC- chimera were also responsive to plastic-bound collagen. When cells were stimulated with collagen in solution, MD.45 cells transduced with scC2Fv/gamma produced IL-2, whereas transduced HCQ6 cells were unresponsive, hence suggesting that the ability of cells transduced with scC2Fv chimeras to respond to soluble collagen correlated with predominant expression of divalent scC2Fv/gamma homodimers, but not monovalent scC2Fv/gamma-CD3zeta or scC2Fv/gammaIC(-)-CD3zeta heterodimers. Of interest, expression of CD3 subunits in hybridomas transduced with scC2Fv chimeras was reduced, resulting in decreased response to cognate Ags.
Systemic sclerosis (SSc) is a complex, immune-mediated rheumatic disease characterised by excessi... more Systemic sclerosis (SSc) is a complex, immune-mediated rheumatic disease characterised by excessive extracellular matrix deposition in the skin and internal organs. B cell infiltration into lesional sites such as the alveolar interstitium and small blood vessels, alongside the production of defined clinically relevant autoantibodies indicates that B cells play a fundamental role in the pathogenesis and development of SSc. This is supported by B cell and fibroblast coculture experiments revealing that B cells directly enhance collagen and extracellular matrix synthesis in fibroblasts. In addition, B cells from SSc patients produce large amounts of profibrotic cytokines such as IL-6 and TGF-b, which interact with other immune and endothelial cells, promoting the profibrotic loop. Furthermore, total B cell counts are increased in SSc patients compared with healthy donors and specific differences can be found in the content of naïve, memory, transitional and regulatory B cell compartments. B cells from SSc patients also show differential expression of activation markers such as CD19 which may shape interactions with other immune mediators such as T follicular helper cells and dendritic cells. The key role of B cells in SSc is further supported by the therapeutic benefit of B cell depletion with rituximab in some patients. It is notable also that B cell signaling is impaired in SSc patients, and this could underpin the failure to induce tolerance in B cells as has been shown in murine models of scleroderma.
consecutive patients attending the outpatient and in-patient departments of the Department of Rhe... more consecutive patients attending the outpatient and in-patient departments of the Department of Rheumatology & Clinical Immunology at our institute, classified as SSc as per the ACR/EULAR 2013 criteria, were included after taking their informed consent. Age and gendermatched healthy controls were included and all enrolled subjects were subjected to a dual energy X-ray absorptiometry (DEXA) scan. Demographic and clinical details of all patients were recorded. Osteoporosis was defined as per the WHO definition of a T score-2.5. The study was approved by the Institutional Review Board and the Institutional Ethics Committee. Results 90 patients with SSc and 45 age and gender-matched healthy controls were included in the study. Confounding factors for bone health, like smoking or alcohol intake, the proportion of post-menopausal women, and past or family history of fragility fractures, were matched between the two groups. The prevalence of osteoporosis among SSc cases was significantly higher than in the healthy controls (34.4% vs. 6.6%, p < 0.001). In univariate analysis, age, low body mass index (BMI), post-menopausal status, diffuse cutaneous phenotype, gastrointestinal involvement, digital ulcers, and pulmonary arterial hypertension were found to be significantly associated with osteoporosis. Multivariate analysis revealed that the presence of osteoporosis was significantly associated with lower BMI, post-menopausal status, and the presence of digital ulcers in patients. Interestingly, glucocorticoid use did not appear to increase the risk of osteoporosis in our cohort. Conclusion Patients with systemic sclerosis and digital ulcers, low BMI, or postmenopausal status are at high risk of developing osteoporosis and therefore should be screened for osteoporosis by DEXA scan to prevent unforeseen fracture-related morbidity and mortality. Glucocorticoids may not be the major culprit as evidenced by this study, while control of disease activity may lead to better bone health status.
To define mechanisms of sustained activation of synovial B lymphocytes in RA, we studied hybridom... more To define mechanisms of sustained activation of synovial B lymphocytes in RA, we studied hybridomas established from the local synovial B cell repertoire of two RA patients for Vκ gene expression and for antigen-binding specificity. The analyses revealed that members of the main Vκ families (I, II and III) were utilized at frequencies consistent with random Vκ gene family use. Furthermore, although the hybridomas expressed genes frequently seen in response to other self- and exogenous antigens, only one VκI- and two of three VκIII-expressing hybridomas exhibited reactivity with self-antigens. Nucleotide sequence analysis revealed that all hybridomas, with the exception of rheumatoid factor (RF)-producing hybridomas, expressed Vκ genes highly related to known germ-line genes (99.3–100% homology) and that diversity was generated by deletions and random nucleotide insertions at the Vκ–Jκ junction. Examination of the few nucleotide changes seen within the Vκ genes revealed a predominance of silent to replacement changes. Moreover, most of these changes can be attributable either to allotypic variations or to limited random nucleotide replacements independent of antigen selection. In contrast, one IgG-RF (B4D8) exhibited predominantly replacement nucleotide changes in the complementarity-determining regions, suggestive of antigen-driven selection. The random expression of immunoglobulin variable region genes with no, or little, evidence of mutation in the synovial B lymphocyte repertoire, including natural polyreactive antibodies, alongside mutated IgG-RF, suggest that both polyclonal activation and antigen-driven responses occur in RA synovia.
The presence of germinal centre-like structures and clonotypic expansion of lymphocytes in RA syn... more The presence of germinal centre-like structures and clonotypic expansion of lymphocytes in RA synovia may indicate a site-specific immune response to local antigens, rather than passively entrapped immune cells, that sustains synovial inflammation. In this study we compare the nature of immunoglobulin light chain variable region gene use in the synovium of RA patients with peripheral B cells to determine the nature of the synovial immune response. Using Vl and Vk gene fingerprinting, which relies on differences in CDR3 length, we demonstrate differences in the pattern of Vl and Vk use and clonotypic expansion of B cells between the synovium and peripheral blood of RA patients. Further, we show that some synovial rearrangements with long CDR3 are selectively expanded. These longer than usual CDR3 were generated by a number of mechanisms including N-additions. However, the observed differences were not uniform in different patients. These observations suggest that local synovial antigens drive significant numbers of T and B lymphocytes selected from an existing repertoire shaped by genetic and environmental factors. Further, the data argue against passive retention of most B cells in the synovium of RA patients.
The NZB mouse is genetically predisposed to develop, at approximately 6 months of age, a spontane... more The NZB mouse is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti‐mouse erythrocyte auto‐antibodies. Molecular analysis of a panel of five anti‐erythrocyte monoclonal antibodies (MoAb) derived from splenocytes of unimmunized NZB mice revealed that these autoantibodies all had functionally rearranged genes from the VH J558 family of immunoglobulin genes with closest homology to germline genes H10 and H30. Owing to clustering of nucleotide differences within the CDRs, compared with the germline, it was concluded that these antibodies were most likely generated by an antigen‐driven mechanism.We report here further molecular analysis of two (4.16.1 and B4.13.2) of the panel of five anti‐mouse erythrocyte producing hybridomas which are apparently clonally related. Nucleotide analysis of the light chain cDNA indicated that both antibodies had closest homology to germline gene Vk24 and use Jk2 gene. Determination of the functional affinities of the MoAb reveal that B4.13.2 (IgG2a) has a &gt; 10‐fold higher affinity for mouse erythrocytes when compared to 4.16.1 (IgGl). This finding supports the view that these two autoantibodies are generated by an antigen‐driven mechanism. The proposed mechanism would involve the selection and expansion of a small population of B‐lymphocytes by antigen leading to isotype switch, somatic mutation and increased affinity. Our data also point to the possibility that some framework residues may be involved in the binding to antigen.
The New Zealand black (NZB) mouse strain is genetically predisposed to develop, at approximately ... more The New Zealand black (NZB) mouse strain is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti-mouse erythrocyte autoantibodies. In order to investigate the molecular mechanisms which lead to anti-mouse erythrocyte autoantibody production we have generated eight anti-mouse erythrocyte MoAbs producing hybridomas from splenocytes of 9-and 12-month-old NZB with spontaneous autoimmune anaemia. IgG2a was the predominant isotype, while IgM, IgGl and IgG2b were each produced by one hybridoma cell line. All anti-mouse erythrocyte MoAbs were characterized for their antigen specificities. None of the MoAbs cross-reacted with ss-or dsDNA or with other species' erythrocytes, with the exception of one MoAb which cross-reacted with rat erythrocytes. None ofthe eight hybridomas was demonstrated to express surface or cytoplasmic CD5, suggesting that they derived from CD5" B lymphocytes. All hybridomas when implanted intraperitoneally into BALB/c mice caused anaemia. In order to define the genetic basis and investigate the molecular mechanisms resulting in pathogenic anti-mouse erythrocyte autoantibody production, the pattern of immunoglobulin variable region gene use has been studied. Five ofthe eight MoAbs whose IgVH genes were sequenced all have functionally rearranged genes from the VH J558 gene family. There is evidence for somatic point mutations in the complementarity-determining regions (CDR) ofthe IgVH genes in all of these five MoAbs when compared with the closest known germline gene. We suggest that these nucleotide sequence changes are likely to reflect selection by an antigen-driven mechanism. Furthermore, the data indicate that pathogenic anti-mouse erythrocytes are not derived from 'natural' autoantibodies.
The specificities of a panel of erythrocyte-reactive MoAbs derived from NZB mice with autoimmune ... more The specificities of a panel of erythrocyte-reactive MoAbs derived from NZB mice with autoimmune haemolytic anaemia (AIHA) were determined by immunoprecipitation and immunoblotting. Of the eight antibodies, two (IgG1 MoAb 105-2H and IgG2a MoAb 34-3C) immunoprecipitated a 105-kD component identified as the erythrocyte anion channel band 3. A similar band was also immunoprecipitated by the IgG2b MoAb 34-2B when used at relatively high concentrations, but none of the remaining hybridoma antibodies precipitated any labelled erythrocyte components. In immunoblotting experiments only 34-2B reacted with band 3, indicating that the epitope recognized by this MoAb is robust and differs from the determinant(s) recognized by 105-2H and 34-3C. The remaining MoAbs to react by immunoblotting were the IgM antibodies IE10 and 4C8, both of which bound to a doublet corresponding to band 4.1 from the internal erythrocyte membrane skeleton. Of the three MoAbs which gave negative results in immunoprecip...
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