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Biomedical subjects

R E Callard

Publications and source records attributed to R E Callard.

At least 19 recordsLinked to original sources
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↗
PubMed1989

Epstein-Barr-virus-transformed lymphoblastoid cell lines derived from patients with X-linked agammaglobulinaemia and Wiskott-Aldrich syndrome: responses to B cell growth and differentiation factors.

Epstein-Barr-virus-transformed B lymphoblastoid cell lines (EBV-transformed LCL) from three patients with X-linked agammaglobulinaemia (XLA), six patients with Wiskott-Aldrich Syndrome (WAS), and seven normal donors, were tested for growth and differentiation in response to human recombinant IL-4, a commercially available, low molecular weight B cell growth factor (BCGFlow), and B cell differentiation factor (BCDF) secreted by the T24 cell line, now known to be IL-6. Proliferation (3H-TdR uptake) by EBV-transformed LCL from both XLA and WAS patients in response to BCGFlow was similar to that obtained with the normal cell lines. In addition, three normal and three WAS, but none of the XLA EBV-transformed LCL, proliferated a little in response to IL-4. All the normal B cell lines secreted IgM, and six out of the seven secreted IgG in response to BCGFlow and BCDF. A similar pattern of response was obtained with the WAS EBV-transformed LCL (6/6 secreted IgM and 4/6 secreted IgG). Several of the normal and WAS EBV-transformed LCL also secreted IgM and IgG in response to IL-4. In contrast, the lines from the XLA patients were abnormal. One secreted large amounts of IgM and two secreted small amounts, but none of the XLA lines secreted IgG constitutively or in response to any of the factors (IL-4, BCDF). The lack of detectable IgG secretion by the XLA lines was probably due to an absence of precommitted IgG B cell precursors transformed by EBV rather than an intrinsic inability to respond to BCGF and BCDF. All of the lines, including those derived from XLA patients, were shown to secrete B cell growth and differentiation factors detected on indicator B cell lines. These results suggest that the abnormal X-linked genes responsible for XLA and WAS do not interfere with B cell responses to B cell growth and differentiation factors.

Agammaglobulinemia↗
PubMed1989

Increased expression of surface IgM but not IgD or IgG on human B cells in response to IL-4.

Surface IgM (sIgM) was increased up to 10 times on human tonsillar B cells activated with IL-4. No change was observed for surface IgD, IgG or IgE. Other activators of human B cells, such as TPA, EBV and anti-IgM resulted in increased expression of the low-affinity receptor for IgE (CD23), but had no effect on sIgM. IL-4 also increased sIgM expression on prolymphocytic leukaemic (PLL) B cells, whereas TPA significantly reduced the level of sIgM. The effect on sIgM thus seems specific for IL-4, and is consistent with the existence of a unique IL-4-dependent B-cell activation pathway. Preincubation with IL-4 did not 'prime' B cells to proliferate in response to subsequent exposure to anti-IgM, and slightly decreased the response to co-stimulation with IL-4 and anti-IgM. The increase in sIgM expression in response to IL-4, therefore, does not seem to be important for proliferation.

B-Lymphocytes↗
PubMed1988

Response of LFA-1-deficient B cells to interleukin 4 (BSF-1) and low molecular weight B cell growth factor (BCGFlow).

T cell-depleted B cells from a patient with LFA-1 deficiency were tested in costimulation assays for responsiveness to recombinant human IL4 (BSF-1) and purified low molecular weight B cell growth factor (BCGFlow). In both cases the response of LFA-1-deficient B cells was comparable with normal controls. Monoclonal antibodies to LFA-1 alpha (CD11a) and beta (CD18) chains were unable to mimic the action of IL4 on normal B cells in costimulation assays with anti-IgM, and did not inhibit normal B cell proliferation in response to IL4 and anti-IgM. Epstein-Barr virus-transformed lymphoblastoid B cell lines (LCL) from normal and LFA-1-deficient donors both responded in proliferation assays to BCGFlow but not IL4. Similarly, both normal and LFA-1-deficient LCL increased IgM secretion in response to BCDF, BCGFlow and, interestingly, IL4. The normal LCL also increased IgG secretion in response to these factors, but no IgG was detected in supernatants from the LFA-1-deficient LCL. These results show that LFA-1 expression is not essential for B cell responses to B cell growth and differentiation factors.

Antibodies, Monoclonal↗
PubMed1988

Interleukin 2 and low molecular weight B cell growth factor are T cell-replacing factors for different subpopulations of human B cells.

Both recombinant human interleukin 2 (rhIL 2) and low molecular weight B cell growth factor (BCGFlow) were shown to be T cell-replacing factors (TRF) in specific antibody responses to influenza virus by human blood and tonsillar B cells. When B cells were separated into high and low-density populations on Percoll gradients at 1.074 kg/l, IL 2 was found to act as a TRF only on the low-density B cells, whereas BCGFlow was a TRF for high-density B cells with a lesser effect on low-density B cells. Both populations of B cells responded well in the presence of T cells. The high-density B cells could not be activated to respond to IL 2 by either IL 1, rhIL 4 or by a CD22 monoclonal antibody known to enhance B cell activation. In contrast, a 24-h preincubation with T cells and antigen appeared to prime high-density B cells to respond to IL 2. These results show that high-density B cells can in fact respond to TRF, and that IL 2 and BCGFlow act on different populations of B cells which may be defined by prior exposure to T cells.

B-Lymphocytes↗
PubMed1988

The marmoset B-lymphoblastoid cell line (B95-8) produces and responds to B-cell growth and differentiation factors: role of shed CD23 (sCD23).

The EBV-producing marmoset B-cell line (B95-8), commonly used as a source of EBV for stimulation and transformation of human B cells, was shown to proliferate in response to supernatants containing human B-cell growth factors (BCGF) derived from PHA-activated T cells or the KG-la cell line, and to a commercial low molecular weight BCGF (BCGFlow), but not to recombinant human IL-4 (rhIL-4). In this respect, B95-8 responded in much the same way as human EBV-transformed lymphoblastoid cell lines (LCL). In contrast, B95-8 did not secrete immunoglobulin in response to B-cell differentiation factor (BCDF) containing supernatants from the KG-la cell line, nor to BCGFlow, or IL-6 obtained from the T24 bladder carcinoma cell line, whereas significant responses were obtained with human EBV-transformed LCL. Both B95-8 and control EBV-transformed human LCL secreted BCGF and BCDF detected with the indicator B-cell lines CESS, L4, and HFB1, but only the human LCL secreted BCGF detectable in co-stimulation assays with TPA-activated tonsillar B cells. Unlike EBV-transformed LCL, B95-8 did not express detectable surface CD23, and did not release into the culture medium soluble CD23 (sCD23) recognized by an EIA for the human molecule. Although not releasing detectable sCD23, B95-8 cells did proliferate in response to purified human sCD23, and were found to be 1000 times more sensitive in this assay than EBV-transformed LCL. This may provide a basis for a sensitive bioassay for sCD23. Unlike EBV-transformed LCL, it seems that in vitro proliferation of B95-8 may involve an autocrine loop which does not depend on CD23.

Animals↗
PubMed1988

B-cell growth and differentiation factors.

The number of recombinant factors which have been shown to regulate B cell activation, proliferation, and differentiation now stands at nine (IL-1, IL-2, IL-4, IL-5, IL-6 (BSF-2), BCGFLOW, IFN-alpha and gamma, and TNF). Several others have been described but are not yet fully characterized or available as recombinant gene products and will not be discussed. Recent work with these factors, especially IL-4, has revealed a remarkable diversity of function. Most of them seem able to act at more than one stage of B cell activation, proliferation and differentiation, thus invalidating the concept of a single unique factor controlling each discrete step of normal B cell responses. In addition, these factors are not B cell specific, but have a wide range of cell types as targets. The functional diversity of these factors has important implications for their potential as therapeutic agents, and for the design of standardization protocols.

Animals↗
PubMed1987

The response of selected human B cell lines to B cell growth and differentiation factors.

Fifteen human B cell lines were tested for their ability to respond to B cell growth and differentiation factors present in phytohemagglutinin-conditioned medium. Five lines responded significantly: CESS showed an increase in IgG production only, HFB1 and BALM1 showed an increase in proliferation only and L4 and BALM4 showed an increase in both IgG production and proliferation. When four of the responding lines (CESS, HFB1, L4 and BALM4) were cultured with human recombinant-derived interleukin 1, interleukin 2, interleukin 4 or interferon-gamma no significant response was seen. CESS, L4 and BALM4 all increased IgG production in response to partially purified B cell growth factor (Cellular Products, Inc., Sera-Lab., Crawley Down, GB) and B cell differentiation factor-containing supernatant from the T24 bladder carcinoma cell line. HFB1, L4 and BALM4 all showed increased tritiated thymidine incorporation in response to purified B cell growth factor but not in response to B cell differentiation factor-containing supernatant. These lines may prove useful in the study of B cell growth and differentiation factors and their receptors.

B-Lymphocytes↗