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R E Callard

Publications and source records attributed to R E Callard.

At least 37 records · Page 2Linked to original sources
PubMed1995

T-lymphocyte activation in steroid-sensitive nephrotic syndrome in childhood.

We undertook a sequential study in 29 children with steroid-sensitive nephrotic syndrome (SSNS) off treatment to seek evidence for T-cell activation in relapse. T-cell subsets and activation markers were analysed using two-colour flow cytometry. Soluble IL2 receptor (sIL2R) was measured in serum and urine by enzyme-linked immunosorbent assay (ELISA). Fifteen children were examined in remission and subsequent relapse (group A) and fourteen remained in remission (group B). In group A the proportion of CD4+ cells expressing the activation marker CD25 (alpha-chain of the IL2 receptor) increased significantly from remission to relapse: CD4+25+ cells rose from 5.6 to 7.0% of total lymphocytes, and from 15.8 to 19.1% of CD4+ lymphocytes (paired t test: P < 0.0005 and < 0.001 respectively). No correlations were found between CD4+25+ cells and plasma albumin or cholesterol concentrations. SIL2R concentration in serum did not change in relapse, but increased significantly in urine from 272 to 592 U/mg creatinine (P < 0.01). No significant difference was found in remission between groups A and B. We conclude that early relapse in SSNS is associated with activation of CD4+ (T-helper) cells which is not secondary due to the nephrotic state itself.

Adolescent↗
PubMed1994

CD40 ligand (CD40L) expression and B cell function in agammaglobulinemia with normal or elevated levels of IgM (HIM). Comparison of X-linked, autosomal recessive, and non-X-linked forms of the disease, and obligate carriers.

Hyper-IgM syndrome is a rare immunodeficiency characterized by low or absent IgG, IgA, and IgE with normal or elevated levels of IgM. It can occur as an acquired or familial disorder with either X-linked or autosomal modes of inheritance. The X-linked form (HIGM1) is a result of mutations in the CD40 ligand (CD40L) gene, but the defect in non-X-linked forms of the disease (HIM) has not been determined. We show here that CD40L expression on activated T cells from non-X-linked patients can be detected by CD40Fc, 5c8 Mab, and anti-TRAP, whereas activated T cells from HIGM1 patients either had no detectable CD40L (Type I), or stained with anti-TRAP but not CD40Fc or 5c8 (Type II). Activated T cells from obligate carriers varied from low to normal expression of CD40L. B cells from HIGM1 and non-X-linked HIM patients proliferated in response to CD40L. Costimulation of B cells from HIGM1, from sporadic HIM, or from non-X-linked HIM patients with CD40L plus IL-2 resulted in some IgM production, but no significant IgG or IgA. Costimulation with CD40L plus IL-10 resulted in significant IgG and/or IgA secretion by B cells from some HIGM1 patients, but consistently failed to stimulate IgG or IgA secretion by B cells from non-X-linked patients. In addition, costimulation with CD40L and IL-4 failed to induce IgE secretion by B cells from one non-X-linked HIM patient, and induced a weak response in another. These results suggest that patients with non-X-linked forms of HIM may have an intrinsic B cell defect preventing heavy chain switching, which is not related to expression of CD40L.

Adolescent↗
PubMed1994

Expression of Bruton's tyrosine kinase protein within the B cell lineage.

Defects in the gene encoding Bruton's tyrosine kinase (Btk), normally expressed in B cells, cause X-linked agammaglobulinemia (XLA). The phenotype of XLA is characterized by a lack of circulating B cells and immunoglobulin. It has been suggested that B cell maturation from the pre-B cell stage to more mature stages is dependent on the appropriate expression of this gene. The Btk mRNA is expressed in B cells and myeloid cells, but protein expression in relation to B cell maturation has not been determined. Moreover, expression of the Btk protein has so far only been investigated in human Epstein-Barr virus-transformed B cell lines, and in murine splenocytes and B cell lines. We have developed an antiserum which recognizes the human Btk protein and shown that normal human tonsillar B cells, peripheral blood monocytes and myeloid cells express the protein, whereas tonsil-derived T cells do not. We also show that the protein is present in early and mature human B cell lines, but is absent in terminally differentiated plasma cell lines. Furthermore, expression is reduced or absent in three B lineage cell lines derived from two patients with defined genetic mutations in Btk and suffering from XLA.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1993

Interleukin 6 is not required for antigen-specific antibody responses by human B cells.

Interleukin-6 (IL-6) is a late-acting differentiation factor for human B cells activated by polyclonal mitogens such as pokeweed mitogen (PWM) and Staphylococcus aureus Cowan strain I, but its role in specific antibody responses has not been established. We show here that IL-6 has no consistent effect on specific antibody responses by tonsillar mononuclear cells (TMC) stimulated with influenza virus. A blocking IL-6 antibody also had no effect on antibody production, suggesting that endogenous IL-6 production was not required. In control experiments, this antibody inhibited PWM-stimulated immunoglobulin secretion and proliferation of the IL-6-dependent B cell line B9. A requirement for IL-6 in responses of unfractionated TMC may have been disguised by the presence of T cells. To overcome this problem, we investigated the effect of IL-6 on specific antibody production by T-depleted B cells stimulated with antigen in the presence of IL-2, which is a T cell replacing factor (TRF) for human B cells. Specific antibody production was restored by IL-2, but not IL-6. Neither IL-6 nor anti-IL-6 antibody had any consistent effect on specific antibody production by purified B cells stimulated with antigen and TRF. These experiments show that IL-6 does not have a significant role in antigen (influenza virus)-specific antibody responses by human B lymphocytes.

Antibodies, Viral↗
PubMed1993

Human immunoglobulin class and IgG subclass regulation: dual action of interleukin-4.

Epstein-Barr virus (EBV) was used as a polyclonal human B cell mitogen to investigate the regulation of immunoglobulin class and IgG subclass responses by interleukin-4 (IL-4). Activation of tonsillar B cells with EBV resulted in an early peak of polyclonal immunoglobulin secretion between days 13 and 14 consisting of IgM, IgA, and IgG1, IgG2, IgG3 and IgG4, but not IgE. Addition of IL-4 to EBV-activated B cells at concentrations of 100 U/ml or greater induced the production of IgE and enhanced IgG4 secretion, but had no effect, or more often inhibited the other isotypes. In contrast, low concentrations of IL-4 (1-5 U/ml) significantly increased the production of IgM, IgA, IgG1, IgG2 and IgG3, but had no effect on IgG4 or IgE. The increase in immunoglobulin secretion obtained with low concentrations of IL-4 was found to occur only with high-density (resting) B cells, suggesting that IL-4 was not functioning simply as a late-acting differentiation factor. Low concentrations of IL-4 significantly increased IgG1, IgG2, IgG3, and IgA production by surface (s) IgM+ (sIgG-/sIgA-) B cells which is consistent with heavy chain switching. In some experiments, however, IL-4 enhanced IgM secretion by sIgM+ B cells, and IgA, IgG1, IgG2, IgG3 by sIgM- B cells, suggesting that it may have an additional B cell differentiation factor activity which was not isotype specific. The different effect of IL-4 at high and low concentrations were similar to those observed in B cell activation experiments, and may be due to the existence of high- and low-affinity IL-4 receptors.

Animals↗
PubMed1993

CD40 ligand and its role in X-linked hyper-IgM syndrome.

CD40 ligand (CD40L) on activated T cells binding to CD40 on B cells is of critical importance for Ig heavy-chain switching and rescue of B cells from apoptosis after somatic mutation in the germinal centre. Mutations in the CD40L gene are now known to cause X-linked hyper-IgM syndrome (HIGM1), an immunodeficiency characterized by the absence of serum IgG, IgA and IgE. In this review, we discuss how basic and clinical immunology have combined to provide major insights into the function of CD40 in T-B cell collaboration.

Antigens, CD↗
PubMed1993

IL-4 regulates the morphology, cytoskeleton, and proliferation of human umbilical vein endothelial cells: relationship between vimentin and CD23.

Many of the vascular endothelial changes associated with inflammation can be induced in vitro by the cytokines tumour necrosis factor, IL-1, and IFN gamma. On the other hand, although IL-4 is a powerful mediator of leucocyte function, its influence on endothelial cells has not yet been fully determined. In this study the effect of IL-4 on human umbilical vein endothelial cells has been investigated. It is shown that IL-4 stimulates DNA synthesis over the first 24 h in culture, followed by dramatic alterations in endothelial cell morphology in which the cobblestone appearance of cells grown to confluence in medium changes to a monolayer composed of islands of tightly packed polygonal cells. The morphological changes induced by IL-4 were accompanied by a reorganization of the intracellular vimentin matrix from a diffuse pattern to a perinuclear concentration observed by staining with vimentin antibodies. Similar patterns of vimentin staining were also observed with an antibody to the low affinity IgE receptor (CD23) normally associated with IL-4 activation of B lymphocytes. Our results suggest that this represents cross reactivity between CD23 and vimentin rather than the appearance of CD23 in endothelial cells. Amino acid sequence comparison between CD23 and vimentin indicated significant homology between these two molecules, including a leucine zipper-like motif. Our results suggest that IL-4 may be an important regulator of endothelial cell morphology and function in inflammation.

Amino Acid Sequence↗
PubMed1992

CD19 regulation of human B cell responses. B cell proliferation and antibody secretion are inhibited or enhanced by ligation of the CD19 surface glycoprotein depending on the stimulating signal used.

The regulation of human B cell proliferation and differentiation by the CD19 surface glycoprotein was investigated. As expected, proliferation induced by costimulation with anti-IgM plus IL-4 or IL-2, or with G28.8 antibody plus IL-4 was inhibited by antibody ligation of CD19. In contrast, proliferation of tonsillar B cells to mitogenic doses of PMA (5 ng/ml) or to EBV were enhanced, and proliferation of B cell lines to BCGF(low) was unaffected. Similarly, specific antibody responses by tonsillar B cells to influenza virus, and Ig secretion by the CESS lymphoblastoid cell line in response to IL-6 were inhibited, whereas polyclonal Ig production in response to EBV was enhanced. These results show that human B cell responses may be inhibited or enhanced by CD19 depending on the stimulating signal used. The difference in response to CD19 ligation did not depend on whether proliferation or differentiation was being measured, or whether stimulation was by surface Ig. In experiments using PMA as a T cell independent mitogen, it was found that ligation of CD19 inhibited proliferation of B cells costimulated with low doses of PMA plus G28.5 (CD40) antibody, but enhanced the response to higher (mitogenic) doses with or without costimulation with G28.5. The change from inhibition to enhancement occurred over a very small increase in PMA dose (0.5-1.0 ng/ml) that corresponded exactly to the lowest dose required for mitogenic activity. Finally, we showed that CD19 ligation inhibited the increase in surface expression of CD23, but not IgM, induced by IL-4, showing that CD19 ligation can have opposed effects on different responses to the same signal. Together our results suggest that CD19 activation of human B cells interacts with other signaling events to enhance or inhibit the subsequent response.

Antibody Formation↗
PubMed1992

Interleukin-4 stimulates immunoglobulin secretion by Epstein-Barr virus (EBV)-activated tonsillar B cells, and by EBV-transformed lymphoblastoid B cell lines without increasing cell division.

Freshly prepared Epstein-Barr virus-transformed B lymphoblastoid cell lines derived from five different donors were tested for their responses to recombinant human interleukin-4 and to low molecular weight B cell growth factor. In the absence of either cytokine, all five lines secreted immunoglobulin of more than one isotype (IgM, IgG, and IgA, but not IgE). Stimulation with interleukin-4 resulted in a significant increase in immunoglobulin secretion, but did not enhance cell division measured by tritiated-thymidine uptake or cell counts. In contrast, low molecular weight B cell growth factor increased both immunoglobulin secretion and cell division. The increase in immunoglobulin secretion stimulated by interleukin-4 occurred for each of the different isotypes (IgM, IgG and IgA) produced by the unstimulated line. No IgE secretion was detected for any of the five lines. It was also found that low (5 units/ml), but not high (100 units/ml), concentrations of interleukin-4 increased IgM, IgG and IgA secretion by tonsillar B cells polyclonally activated with Epstein-Barr virus. Again, no IgE was detected at any time. These results suggest that interleukin-4 can function as a late-acting B cell differentiation factor as well as a growth factor for human B cells.

Animals↗
PubMed1991

Inhibition of B cell proliferation with anti-CD19 monoclonal antibodies: anti-CD19 antibodies do not interfere with early signaling events triggered by anti-IgM or interleukin 4.

The 95-kDa antigen recognized by the anti-CD19 panel of monoclonal antibodies is found on the surface of most cells of the B cell lineage. Anti-CD19 antibodies inhibit B cell proliferation in response to anti-Ig plus interleukin 4 (IL4), but enhance the response to mitogenic concentrations of either phorbol 12-myristate 13-acetate (PMA) or Epstein-Barr virus. This dichotomy in the effect of anti-CD19 antibodies suggested that the inhibitory action may be directed at the transmembrane signaling pathways utilized by anti-IgM and IL4. To investigate this hypothesis, an attempt was made to determine the mechanism of signal transduction utilized by the CD19 antigen, and elucidate its effect on transmembrane signaling invoked by anti-immunoglobulin and IL4. Binding of anti-CD19 antibody to B cells did not promote activation of either the phosphoinositide or cAMP signaling pathways. In addition, anti-CD19 antibody did not inhibit phosphatidylinositol bisphosphate (PIP2) hydrolysis induced by anti-IgM or IL4, nor did it interfere with cAMP induction by IL4. We also found that anti-CD19 antibody inhibited PMA plus calcium ionophore-induced B cell proliferation. This evidence indicates that anti-CD19 mAb interrupts the signaling cascade at a point distal to receptor-mediated breakdown of PIP2 and/or activation of adenyl cyclase. This conclusion was fully consistent with experiments in which anti-CD19 antibody was shown to inhibit DNA but not RNA synthesis, and the observation that anti-CD19 antibody must be present between 6 h and 20 h after the initiation of the culture suggesting that anti-CD19 mAb exerts its inhibitory effect in late G0 or G1, after the initial signaling events.

Antibodies, Anti-Idiotypic↗
PubMed1991

The role of interleukin 4 in specific antibody responses by human B cells.

This study was designed to investigate the requirement for interleukin 4 (IL-4) in specific antibody responses by human lymphocytes. Addition of IL-4 to antigen (influenza virus)-stimulated cultures of tonsillar mononuclear cells was found to suppress specific antibody production significantly at doses as low as 10 units/ml. Specific immunoglobulin (IgG), IgA, and IgM antibodies were all equally inhibited by IL-4. Inhibition of the antibody response with IL-4 was completely abrogated by an IL-4 blocking antibody showing that the effect was specific for IL-4. It was also found that anti-IL-4 did not inhibit specific antibody production, showing that IL-4 was not required for responses to antigen. In contrast, significant inhibition was obtained with anti-Tac, indicating an important role for IL-2. In the absence of T helper cells antibody responses to influenza virus were completely restored with T cell replacing factor [TRF; IL-2 or low-molecular-weight B cell growth factor (BCGFlow)], but not with IL-4. In fact, IL-4 significantly suppressed the antibody response obtained when either IL-2 or BCGFlow was used as a TRF. Addition of IL-4 at different times after in vitro stimulation with antigen and IL-2 showed that the inhibitory activity of IL-4 was maximal during the first 3 days of culture and was lost by day 4. IL-4 therefore seems to inhibit an early activation event (possibly dependent on IL-2 or BCGFlow), or B cell proliferation essential for specific responses to antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1991

Independent regulation of interleukin 4 (IL-4)-induced expression of human B cell surface CD23 and IgM: functional evidence for two IL-4 receptors.

Activation of human B cells with interleukin 4 (IL-4) is known to result in increased expression of CD23 (the low-affinity receptor for IgE) and sIgM. However, whereas CD23 expression is increased by several B cell mitogens, including phorbol 12-myristate 13-acetate, Epstein-Barr virus, anti-immunoglobulin (Ig), and IL-4, surface IgM (sIgM) expression is increased only with IL-4, suggesting that expression of each surface antigen is regulated independently. This was confirmed in three different ways. First, in dose-response experiments, it was shown that 10 times the concentration of IL-4 was required for CD23 than for sIgM expression. Similar or even higher concentrations of IL-4 were required for proliferation. In fact, optimal sIgM expression was obtained in some experiments with concentrations of IL-4 (1-5 units/ml) which had little or no effect on either CD23 expression or B cell proliferation. Secondly, IL-4 is known to activate the phosphatidyl inositol pathway in human B cells followed 8-10 min later by an increase in cAMP. Pharmacologically mimicking this pathway by brief exposure of resting B cells to phorbol dibutyrate plus ionomycin followed 10 min later with dibutyryl cAMP resulted in an increase in expression of CD23 but not sIgM. Thirdly, CD19 monoclonal antibody, which inhibits B cell proliferation in response to IL-4 plus anti-Ig, was found to inhibit IL-4-induced CD23 but not sIgM expression. These results show that CD23 and sIgM expression are regulated independently and are consistent with the existence of two separate signal transduction pathways stimulated by IL-4, which may be coupled to distinct IL-4 receptors.

Antigens, CD19↗
PubMed1991

Activation of human B cells through the CD19 surface antigen results in homotypic adhesion by LFA-1-dependent and -independent mechanisms.

Addition of CD19 monoclonal antibodies (mAb) to highly purified tonsillar B cells resulted in homotypic adhesion and the formation of cell clusters. This response was completely blocked by antibody to LFA-1, indicating an LFA-1-dependent adhesion mechanism. In contrast, aggregate formation by B cells activated with phorbol myristate acetate (PMA) was only partially inhibited by anti-LFA-1 antibody, and those formed in response to PMA plus CD19 antibody were not inhibited at all, suggesting aggregation of activated B cells stimulated with CD19 antibody was LFA-1 independent. This was confirmed with B-cell lines. The pre-B-cell line Nalm-6 formed aggregates in response to CD19 antibody which were not inhibited with anti-LFA-1. In addition, CD19 antibody induced aggregate formation by an Epstein-Barr virus (EBV)-transformed B-cell line derived from an LFA-1-deficient donor. These results suggest that different adhesion molecules may operate at different stages of B-cell activation, and that CD19 may be important in cell-cell interactions involved in regulation of antibody responses.

Antibodies, Monoclonal↗
PubMed1990

Interleukin 4 activates human B lymphocytes via transient inositol lipid hydrolysis and delayed cyclic adenosine monophosphate generation.

We report from three independent centers that, in human tonsillar B lymphocytes, human IL4 switches on a series of second messenger changes, the precise sequence of which constitutes a novel signal transduction cascade. It involves an immediate and transient elevation of inositol 1,4,5-trisphosphate and Ca2+ levels. This is followed several minutes later by a sustained rise in cellular cyclic adenosine monophosphate concentration, the triggering of which involves both the Ca2+ rise and an additional, as yet unidentified, IL4-generated signal. Both the products of the initial inositol lipid hydrolysis and the delayed cyclic adenosine monophosphate accumulation are essential for the later induction of CD23 expression, a major phenotypic change promoted in these cells by IL4. The striking contrast between these findings and those that have been observed for the IL4 triggering of murine B cells is discussed.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1989

Human T cell-replacing factor(s): a comparison of recombinant and purified human B cell growth and differentiation factors.

Conditioned medium from phytohemagglutinin-activated T cells contains T cell-replacing factor(s) (TRF) able to restore specific antibody responses by human blood or tonsillar B cells which have been thoroughly depleted of T cells. Of twelve recombinant cytokines tested as possible candidates for TRF in conditioned media, namely human recombinant interleukin (hrIL) 1 alpha and beta, hrIL2, hrIL3, hrIL4, hrIL5, hrIL6, hrIFN-alpha and -gamma, hr granulocyte macrophage colony-stimulating factor (hrGM-CSF) and tumor necrosis factor (hr TNF)-alpha and -beta only IL2 was found to have TRF activity. In addition, a semi-purified low molecular weight B cell growth factor (BCGFlow) also had TRF activity. As the commercially available BCGFlow is known to contain low concentrations of IL2, IFN-gamma, TNF and GM-CSF as impurities, it was important to exclude these as being responsible for the TRF activity. At the concentrations present in BCGFlow (less than 0.2 U/ml), IL2 was not active in the TRF assay. In contrast, a combination of IL2 (0.2 U/ml), IFN-gamma (50 U/ml), TNF-alpha (50 U/ml) and TNF-beta (100 U/ml) did have TRF activity suggesting that B cells could be made to respond to low doses of IL2 by the presence of other cytokines. Although this finding raises important questions about the nature of TRF in conditioned medium, the TRF activity of BCGFlow was unlikely to be due to such a synergistic combination of cytokines for the following reasons. First, in several experiments, responses were obtained with BCGFlow, but not with IL2 or combinations of IL2 with IFN and TNF. Second, antibody to IL2 was found to inhibit the TRF activity of IL2 but not of BCGFlow. Taken together these findings show that two distinct cytokines (IL2 and BCGFlow) are TRF for human B cells. However, some combinations of cytokines can also have TRF activity underlining the complexities which can arise from working with semi-purified rather than recombinant factors.

Antibody Formation↗
PubMed1989

Cytokine regulation of B-cell growth and differentiation.

In the last few years, more than ten soluble factors (cytokines) important for regulating B cell growth and differentiation have been identified and their genes cloned. These factors are now known to influence each separate stage of normal B cell responses, namely the activation of quiescent B cells, their proliferation and differentiation into antibody secreting cells and the regulation of immunoglobulin class and subclass production. An important feature of all of these factors is their lack of target cell specificity. Each one has multiple activities both on B cells and on a range of other cell types, often in synergy with other factors. How their multiple functions are controlled in vivo is only now beginning to be understood, opening the way to the use of factors--or more probably, specific inhibitors--for the treatment of a variety of immunological diseases including allergy, autoimmunity, and possibly some forms of B cell malignancies.

Animals↗