Biomedical subjects

P M Lydyard

Publications and source records attributed to P M Lydyard.

At least 19 recordsLinked to original sources

PubMed2000

Induction of tumor necrosis factor alpha production by adhered human monocytes: a key role for Fcgamma receptor type IIIa in rheumatoid arthritis.

OBJECTIVE: Small IgG rheumatoid factor immune complexes may provide the trigger for macrophage-derived tumor necrosis factor alpha (TNFalpha) production in rheumatoid arthritis. Immune complexes may bind to any of 3 IgG Fc receptors (FcgammaR). Therefore, the ability of monocyte-derived macrophages to produce TNFalpha was examined following ligation of each of the 3 human FcgammaR, using murine monoclonal antibodies (mAb) to each receptor as a model for small immune complexes. METHODS: Adhered human monocytes expressing all 3 FcgammaR were incubated with murine anti-FcgammaR mAb directed against FcgammaRI, FcgammaRII, or FcgammaRIII. Supernatants were collected at various time points and tested for the presence of TNFalpha and interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay. RESULTS: The anti-FcgammaRIII mAb induced adhered human monocytes to release TNFalpha. However, F(ab)2 and Fab fragments of the anti-FcgammaRIII mAb failed to induce TNFalpha production. TNFalpha was undetectable following incubation with the anti-FcgammaRI or anti-FcgammaRII mAb. Furthermore, blocking FcgammaRI or FcgammaRII had no effect on the levels of TNFalpha released in response to the anti-FcgammaRIII mAb. Of the 3 anti-FcgammaR mAb, only anti-FcgammaRIII induced IL-1alpha production from adhered human monocytes, and this was inhibited by the presence of a neutralizing anti-TNFalpha mAb. CONCLUSION: This study suggests a dominant role for FcgammaRIIIA in the induction of both TNFalpha and IL-1alpha production by human macrophages in rheumatoid arthritis following receptor ligation by small immune complexes. The signaling of TNFalpha production may require the ligation of either 3 FcgammaRIIIA receptors or only 2 FcgammaRIIIA receptors, where one interaction must involve binding via an Fc domain. In addition, IL-1alpha production following FcgammaRIIIA ligation appears to be dependent on the presence of TNFalpha.

Antibodies, Monoclonal↗

PubMed2000

Human monoclonal anti-phospholipid antibodies selectively bind to membrane phospholipid and beta2-glycoprotein I (beta2-GPI) on apoptotic cells.

The ability of an anti-phospholipid (LJ1) and an anti-beta2-GPI (RSP-57) human MoAb to bind to apoptotic but not viable cells was demonstrated in this study. Both MoAbs were derived from patients with systemic lupus erythematosus and anti-phospholipid antibody syndrome. The parallel analysis of the specificity and affinity of four anti-phospholipid human MoAbs suggests that the binding of LJ1 MoAb to apoptotic cells is a specific property of this MoAb. RSP-57 MoAb recognizes apoptotic cells through beta2-GPI which becomes available for binding after the interaction with negatively charged phospholipids. This observation provides evidence that the binding of human anti-phospholipid antibodies to apoptotic cells occurs in both a beta2-GPI-dependent and independent way and involves a restricted group of epitopes. The finding that LJ1 and RSP-57 MoAbs bind apoptotic cells underlines the property of these MoAbs to act as cell membrane markers of apoptosis. Major pathological implications derive from the observation that LJ1 and RSP-57 MoAbs recognize epitopes expressed on 'early' apoptotic cells. The interference with the in vivo clearance and processing of apoptotic cells is a potential pathogenic mechanism of these antibodies.

Animals↗

PubMed2000

Decreased expression of FcgammaRIII (CD16) by gammadelta T cells in patients with rheumatoid arthritis.

Some gammadelta T cells express a receptor for the Fc portion of immunoglobulin G (FcgammaRIII - CD16). The relevance of this Fc receptor to gammadelta T-cell function is at present unclear. Our previous studies have shown that gammadelta T cells express activation markers in patients with rheumatoid arthritis (RA). In this study we have examined the relative proportions of CD16+ gammadelta T cells in the blood and synovial fluid of these patients compared with control blood. CD16+ gammadelta T cells from RA patients were significantly reduced in synovial fluid compared with the circulation. That this was due to blocking of antibody binding to CD16 was unlikely as treatment of blood gammadelta T cells with RA synovial fluid (known to contain immune complexes) failed to alter expression of CD16. Treatment of blood gammadelta T cells with phytohaemagglutinin in vitro, resulted in a time-dependent decrease in expression of CD16, with a concomitant increase in expression of human leucocyte antigen-DR, at the single cell level. We conclude that expression of CD16 by gammadelta T cells is lost in the synovial compartment as the result of activation.

Arthritis, Rheumatoid↗

PubMed2000

CD5-positive B cells at the crossroads of B cell malignancy and nonorgan-specific autoimmunity.

The CD5 glycoprotein is detectable on a minute fraction of circulating B cells. The number of CD5+ B cells (B1 cells) is increased in patients with autoimmune disorders and these cells, similar to those leading to chronic lymphocytic leukemia (CLL), may be induced to produce multispecific autoantibodies. CD5 is associated with the B cell antigen receptor, and the induction of apoptosis depends on the activation state of the cells. Defective regulation of this phenomenon might result in the production of autoantibodies and/or the development of CD5+ B cell tumors. We have proposed that there may be different B1 populations in man, which depend on the consequence of CD5 ligation on their surface: 'natural' or 'classical' CD5+ B cells, and 'induced' CD5+ B cells.

Apoptosis↗

PubMed1999

CD5 expression in human B-cell populations.

The origin of CD5+ B cells remains controversial. The differential response to ligation of CD5 resulting in apoptosis or proliferation provides insight into its roles in distinct human B cells. Here, Pierre Youinou, Christophe Jamin and Peter Lydyard review current knowledge of B-1 and B-2 cells, and propose that CD5 has different functions when expressed by different B-cell subpopulations.

Animals↗

PubMed1999

The effect on immunoglobulin glycosylation of altering in vivo production of immunoglobulin G.

The effect on murine immunoglobulin G (IgG) glycosylation of altering IgG production in vivo was assessed in interleukin (IL)-6 transgenic and CD4 knockout mice. C57BL/6 mice carrying the IL-6 transgene showed increased levels of circulating IgG. This was associated with decreased levels of galactose on the IgG oligosaccharides. No decrease in beta4-galactosyltransferase mRNA or in enzyme activity was seen in IL-6 transgenic mice. MRL-lpr/lpr mice normally have elevated levels of circulating IgG, again accompanied by decreased levels of IgG galactose. Disruption of the CD4 gene in MRL-lpr/lpr mice led to a substantial decrease in the concentration of circulating IgG, but IgG galactose levels remained low. Thus, an enforced decrease in IgG levels in the lymphoproliferative MRL-lpr/lpr mice did not alter the percentage of agalactosyl IgG in these mice, suggesting that agalactosyl IgG production is not simply caused by excessive IgG synthesis leading to an insufficient transit time in the trans-Golgi, but rather to a molecular defect in the interaction between galactosyltransferase and the immunoglobulin heavy chain.

Animals↗

PubMed1999

CD5 B cells and B-cell malignancies.

Over the past year, progress has been made in understanding of the physiology and disease associations of CD5+ (B1) B cells, although their exact role in pathogenesis remains unclear. Earlier studies on the negative function of CD5 within the B-cell receptor complex have been substantiated, and it seems likely that soon the signaling pathways used by this coreceptor will be elucidated. Progress in diagnosis, physiology, and etiopathogenesis of CD5+ malignancies has been made, particularly in B-cell chronic lymphocytic leukemia. The low-level expression of surface immunoglobulin has been explained by the mutations that occur in the associated CD79b. Two new potential tumor-suppressor genes have been identified in the hot spot of chromosome 13q, which provides an exciting step forward in understanding of the etiopathogenesis of some B-cell chronic lymphocytic leukemia. Activated signal transducers for activation of transcription factors molecules have been shown to be phosphorylated on different amino acids in B1 and chronic lymphocytic leukemia tumors, although the significance of this is, as yet, unclear. Finally, aberrant expression of CD40L by chronic lymphocytic leukemia T cells may contribute to the immunodeficiency that develops in these patients.

Apoptosis↗

PubMed1998

Ligation of CD5 on resting B cells, but not on resting T cells, results in apoptosis.

The CD5 molecule is expressed by a B cell subset. We have demonstrated that resting B cells do not proliferate in response to CD5 ligation, whereas cells preactivated with anti-IgM and IL-2 do so. Here, we specifically studied the effects of anti-CD5 and anti-IgM on apoptosis of CD5+ B cells. Both ligation of CD5 or of surface IgM (sIgM) resulted in apoptosis. This started earlier following ligation of CD5 than with sIgM, and both responses were time dependent. CD5-induced apoptosis was independent of the epitope recognized or the way the antibody was presented to the B cells. CD5+ B cells were more sensitive to IgM-induced apoptosis than CD5 B cells. Engagement of CD5 or CD3 expressed by T cells failed to induce apoptosis. Our data indicate differences in the function of CD5 molecules on tonsillar B cells, compared with blood T cells and suggest that cross-linking CD5 on B cell activates specific pathways responsible for apoptosis.

Apoptosis↗

PubMed1998

Antibody recognition of epitopes on wild-type and mutant beta-(1-->4)-galactosyltransferase-1.

The epitopes present on beta-(1-->4)-galactosyltransferase-1 (beta 4Gal-T1) have been explored using a panel of monoclonal antibodies (mAbs) raised against the soluble form of the human enzyme. Reactivity of the antibodies with site-specific and truncated mutants of human beta 4Gal-T1 suggests the presence of a major immunogenic epitope cluster consisting of four epitopes within the stem region and mapping between amino acids 42 and 115. The catalytic activity of the enzyme is increased in the presence of stem region-specific antibody. Two of the epitopes were further localized to a region between amino acids 42 and 77, sequences which are not shared with the recently cloned beta 4Gal-T2 and beta 4Gal-T3 enzymes. An epitope located close to or within the catalytic domain is also identified, and the mAb to this region binds synergistically with antibodies to the stem region.

Antibodies, Monoclonal↗

PubMed1998

B lymphocyte galactosyltransferase protein levels in normal individuals and in patients with rheumatoid arthritis.

We have quantified the level of beta4-galactosyltransferase protein in human B lymphocytes using an ELISA-based assay. Between 1-10ng of beta4-galactosyltransferase was detected per mg total cellular protein, indicating that this enzyme constitutes <0.001% of B lymphocyte cellular protein. Akin to previous studies, individuals with rheumatoid arthritis exhibited reduced lymphocytic galactosyltransferase enzyme activity compared with normal controls when using ovalbumin as the acceptor substrate. The levels of enzyme protein present in B lymphocytes from patients with rheumatoid arthritis was, however, not reduced suggesting that the B lymphocyte galactosyltransferase catalytic activity may be regulated post-translationally.

Aged↗

PubMed1998

The effect on IgG glycosylation of altering beta1, 4-galactosyltransferase-1 activity in B cells.

An absence of galactose on the N-linked oligosaccharides of immunoglobulin G (IgG) has been shown to affect the functional activity of the antibody molecule. In patients with rheumatoid arthritis there is an increased proportion of IgG which lacks galactose and correspondingly lower levels of beta1, 4-galactosyltransferase (beta4Gal-T) activity. The recent demonstration of several expressed beta4Gal-T genes in man raises the possibility that the enzyme responsible for the decreased IgG galactose is not the "classical" beta4Gal-T (beta4Gal-T1). To directly address the question of whether reduced beta4Gal-T1 would lead to reduced IgG galactose, the level of beta4Gal-T1 in a human IgG-secreting B cell line was specifically altered using stable transfection with sense (SpcDNA3-Gal-T1) or antisense (ASpcDNA3-Gal-T1) human beta4Gal-T1 cDNA. SpcDNA3-Gal-T1 B cell transfectants expressed up to a 2.5-fold higher level of beta4Gal-T enzyme activity for the exogenous neoglycoconjugate acceptor GlcNAc-pITC-BSA than did ASpcDNA3-Gal-T1 transfectants. Flow cytometric analysis with Ricinus communis agglutinin I (RCAI) revealed an overall greater number of Galbeta1,4GlcNAc structures in the fixed and permeabilized SpcDNA3-Gal-T1 B cell transfectants compared with the ASpcDNA3-Gal-T1 transfectants. Moreover, there was increased galactosylation of IgG secreted from the SpcDNA3-Gal-T1 transfectants relative to the ASpcDNA3-Gal-T1 B cell transfectants. Alteration of the level of the "classical" beta4Gal-T (beta4Gal-T1) in B cells therefore affects IgG glycosylation.

B-Lymphocytes↗

PubMed1997

The stability of lymphocytic beta 1,4-galactosyltransferase expression during pregnancy and lactation.

The authors used murine pregnancy as a model to investigate the regulation of IgG glycosylation. Pregnancy is associated with decreased levels of circulating IgG. The oligosaccharides on this IgG from late (day 15), but not early (day 8), pregnant Balb/c mice exhibited increased levels of terminal galactose. The levels remained elevated 8 days post-partum in lactating mice. Nonetheless, splenic beta 1, 4-GalTase mRNA and enzyme activity remained relatively constant throughout pregnancy and into lactation. This was in contrast to a pregnancy-associated increase in mammary gland beta 1,4-GalTase mRNA. Thus the increased IgG galactose levels seen in pregnancy are regulated by mechanisms which are independent of transcriptional control of beta 1,4-GalTase expression.

Animals↗

PubMed1997

Modulation of CD72 by ligation of B cell receptor complex molecules on CD5+ B cells.

B cells expressing CD5 also carry its ligand, CD72. As an approach to understanding the role of CD5 and CD72 on B cells, we have examined the association of CD72 with CD5 and slgM by modulation/co-modulation and capping/co-capping following ligation of these surface molecules with specific antibodies. Modulation and co-modulation were measured after 24 h, whilst capping was measured after 1 h. CD5 and slgM co-modulated each other, CD72 co-modulated with slgM and CD5, but anti-CD72 did not affect either slgM or CD5. CD5 and slgM co-capped each other, whilst CD72 failed to co-cap with either slgM or CD5. The CD5-induced co-modulation of CD72 was partially blocked by specific protein tyrosine kinase inhibitor, but not the slgM-induced co-modulation, Protein kinase C (PKC) inhibitors abrogated the anti-mu- but not the anti-CD5-triggered modulation of CD72, whereas PKC activators prevented the CD5- but not the slgM-induced 24 h modulation of CD72. None of these drugs was able to modify the anti-CD72-induced modulation of CD72. Our data suggest that CD5 is physically associated with slgM in the B cell receptor complex but not with CD72. Furthermore, from the effect of drugs on modulation, there appears to be different associations of CD72 with slgM and CD5. These two pathways differed in some respects, consistent with a co-stimulatory function of CD72 and CD5 in B cell activation.

Antigenic Modulation↗

PubMed1997

Lack of NK cells in lupus patients with renal involvement.

We have previously shown that patients with SLE have significantly lower percentages and absolute numbers of NK(CD3-/CD16+56) cells in their peripheral blood compared with normals. Patients with active disease had very low levels of NK cells and the reduction was also associated with patients who had renal involvement. We have now performed a serial study immunophenotyping 11 patients with SLE and renal involvement using dual colour immunofluorescence and flow cytometry. Patients were tested every three months on an average of three occasions. As a control, nine SLE patients without renal involvement were immunophenotyped for similar intervals; 11 normal controls were also tested. Major lymphocyte subsets (T, B and NK) remained very stable during serial bleeds. However, the NK cell populations were decreased significantly in patients with renal involvement both as percentages (5 +/- 6 vs 9 +/- 5, P < 0.0001) and absolute counts (75 +/- 108 vs 109 +/- 52, P < 0.001) in comparison to non-renal patients. Analysis of disease activity using BILAG score showed an inverse correlation between renal system activity and percentage and absolute number of NK cells (P < 0.002 and 0.01, respectively). In this study we have also analysed a CD8 T cell subset which we have not studied before. We have found a significantly increased percentage of CD38+CD8+ T cells(activated CD8 subset) in patients with SLE in comparison to normal controls. We did not find any association with the CD38+CD8+ T cells and disease activity as measured by BILAG or renal involvement. NK cells are important factors in immunity against virus infections and tumour cells. CD38+CD8+T cells are increased in viral infections. We speculate that the lack of NK cells in SLE patients might have an association with increased CD38 expression.

Adult↗

PubMed1996

Anti-CD5 extends the proliferative response of human CD5+ B cells activated with anti-IgM and interleukin-2.

The CD5 T cell glycoprotein which is expressed by a subset of B cells has been shown to be involved in T cell activation and proliferation. No similar studies, to date, have addressed the role of CD5 on the B cell subset. CD5+ and CD5- B cells were sorted and stimulated with anti-CD5 monoclonal antibody (mAb) in vitro. The activation and proliferative responses of these two populations, as measured by analysis of proliferation marker, did not differ following anti-mu and interleukin (IL)-2 stimulation. The addition of anti-CD5 did not change the responsiveness of such activated CD5+ B cells but resulted in a decrease in CD25 expression. Pre-activation of B cells with phorbol 12-myristate 13-acetate, which increased CD5 expression, failed to alter the proliferative response of CD5+ B cells to anti-mu and IL-2 with or without addition of anti-CD5 mAb. Anti-mu and IL-2 treatment of CD5+ cells resulted in optimal proliferation measured at day 3 which decreased by day 6. However, addition of anti-CD5 mAb at day 3 prevented this decline in proliferative response. This dose-dependent effect was observed only when the anti-CD5 mAb was presented to the B cells in cross-linked form. Co-stimulation of CD5 did not lower the threshold of antigen to which the B cells responded. Taken together, these data support a functional role for CD5 on B cells acting as an accessory signal, following their primary activation through the B cell receptor complex and highlight differences in the role of CD5 associated with the T cell receptor complex.

Antibodies, Anti-Idiotypic↗

PubMed1996

Differential expression of Fc receptors for IgG by monocytes and granulocytes from neonates and adults.

The immature neonatal immune system is thought to result in increased risk of infection. Receptors for the Fc moiety of IgG (Fc gamma R) are important in antibody-mediated clearance of microbes by granulocytes and monocytes/macrophages. As an approach to understanding their role in neonatal life, we have compared the constitutive expression of the three Fc receptors--Fc gamma RI (CD64), Fc gamma RII (CD32) and Fc gamma RIII (CD16)--by neonatal and adult blood monocytes and granulocytes using quantitative immunofluorescence by flow cytometry. Our results confirm that there is a small subpopulation of Fc gamma RIII-positive monocytes in adult blood, and furthermore show that this is absent or at a low percentage in cord blood samples. However, the main population of cord blood monocytes expresses low, but significantly higher levels of Fc gamma RIII than adult monocytes. No differences were seen in the quantitative expression of Fc gamma RI and Fc gamma RII. Neonatal granulocytes expressed significantly higher levels of both Fc gamma RI and Fc gamma RII but significantly lower levels of Fc gamma RIII. The data are discussed in terms of the possible role of cytokines and susceptibility to infection.

Adult↗

PubMed1996

Diverse antigen specificity of erythrocyte-reactive monoclonal autoantibodies from NZB mice.

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 immunoprecipitation and immunoblotting, the IgM antibodies 103-7E and 106-10E reacted poorly with intact erythrocytes by flow cytometry, but the IgG1 antibody 31-9D bound well. ELISAs demonstrated that all four IgM MoAbs are polyreactive, since they bound to histones from a panel of nuclear antigens, and additionally 103-7E reacted with phosphatidyl choline. It is concluded that band 3 is an important autoantigen in NZB AIHA. However, since 3/5 haemolytic MoAbs failed to participate this antigen, either these antibodies represent minor components of the total autoantibody response, or responses to diverse possibly non-protein surface antigens also contribute to the pathogenesis of the disease.

Anemia, Hemolytic, Autoimmune↗

PubMed1996

Agalactosyl IgG and beta-1,4-galactosyltransferase gene expression in rheumatoid arthritis patients and in the arthritis-prone MRL lpr/lpr mouse.

Reduced galactosylation of immunoglobulin G (IgG) is well documented in rheumatoid arthritis (RA), but the reason for this defect is still unknown. There is some evidence supporting a defect in the biosynthetic pathway, and a reduction in the level of beta-1,4-galactosyltransferase (beta-1,4-GalTase) enzyme activity. Since glycosyltransferases are, in general, regulated at the level of transcription, we have measured the level of beta-1,4-GalTase gene expression in B cells from patients with RA and normal control individuals. We found no significant difference in mRNA levels for the transferase in these two groups (P > 0.7). MRL/Mp-lpr/lpr (MRL-lpr) mice develop a spontaneous arthritis with increased levels of agalactosyl IgG (G0). In spite of a significant reduction in the level of beta-1,4-GalTase mRNA in total spleen lymphocytes from MRL-lpr mice compared with the congenic MRL/Mp-(+/+) (MRL-(+/+) mice and with CBA/Ca mice, we found comparable levels of the beta-1,4-GalTase mRNA in purified B cells from both spleen and lymph nodes of the three strains. Amongst the lymphoid compartments examined, the spleen and peripheral blood were found to be the major contributors of G0 in MRL-lpr mice. These data indicate that in neither human RA, nor in an animal model of this disease, is reduced IgG galactosylation caused by impaired expression of the beta-1,4-GalTase gene in B lymphocytes. Furthermore, splenic B cells, which have normal levels of beta-1,4-GalTase mRNA, appear to be a major source of G0 in MRL-lpr mice.

Animals↗