Biomedical subjects

R J Armitage

Publications and source records attributed to R J Armitage.

At least 19 recordsLinked to original sources

PubMed1992

Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion.

Signaling through the cell surface molecule, CD40, is known to play an important role in the proliferation and differentiation of B lymphocytes. Using the thymoma cell line EL4, we recently identified and cloned a cDNA encoding a murine ligand for the CD40 molecule (mCD40-L) and showed that it has biological activity in vitro. A cDNA encoding a human homologue of the mCD40-L was isolated using crosshybridization techniques from an activated peripheral blood T cell library. The predicted amino acid sequence indicates that this human ligand for CD40 (hCD40-L) is a 261 amino acid type II membrane protein that exhibits 78% amino acid identity with its murine counterpart. Northern blot and FACS analyses suggest that the hCD40-L is restricted in its expression to T lymphocytes, and that it is most abundant on the CD4+ T cell subpopulation. Cells transfected with hCD40-L caused the proliferation of human tonsil B cells in the absence of costimuli and, in the presence of interleukin 4, induced immunoglobulin E secretion from purified human B cells. A comparison of the efficacy of the hCD40-L and mCD40-L in these assays is presented.

Amino Acid Sequence↗

PubMed1992

Regulation of human monocyte cell-surface and soluble CD23 (Fc epsilon RII) by granulocyte-macrophage colony-stimulating factor and IL-3.

We have investigated the regulation of expression of cell-surface and soluble CD23 (sCD23) by purified human peripheral blood monocytes and in cultures of human whole blood. IL-3, IL-4, and GM-CSF were found to markedly enhance the expression of CD23 on the surface of elutriated monocytes and to increase levels of sCD23 in monocyte-culture supernatants. The induction of CD23 expression by monocytes was confirmed at the mRNA level by Northern blot analysis. The ability of GM-CSF, IL-3, or IL-4 to induce cell-surface CD23 on monocytes was inhibited by specific neutralizing antibodies to the corresponding cytokine. IL-3 and GM-CSF induced maximal surface CD23 expression on monocytes by 24 to 48 h, followed by a slight decline at 72 and 96 h. In contrast, IL-4 induced a progressive increase in monocyte CD23 expression that reached a maximum at approximately 72 h. IL-4, GM-CSF, and IFN-gamma increased both surface and soluble CD23 expression by the monocytic cell line U937, whereas IL-3 had no effect. The plasma from fresh human whole blood or nonstimulated whole blood cultured for 24 to 48 h contained detectable sCD23, and addition of IL-3, IL-4, or GM-CSF to these cultures resulted in increased levels of this molecule. Two-color flow cytometry revealed that IL-3, but not GM-CSF, also enhanced CD23 expression by B cells enriched from PBMC, although the effect of IL-3 was weak in comparison with that of IL-4. These findings may have important implications for the in vivo therapeutic use of these cytokines.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1992

Soluble forms of CD40 inhibit biologic responses of human B cells.

We have expressed the CD40 surface Ag as both a soluble 28-kDa molecule and a 57-kDa Fc fusion protein containing the human IgG1 Fc region. Soluble CD40 and the Fc fusion protein inhibited the proliferative response of anti-IgM-activated human B cells to the CD40 mAb G28-5. Similarly, G28-5- and IL-4-induced IgE secretion from PBMC depleted of T cells was effectively blocked by both forms of soluble CD40. Although the soluble constructs of CD40 had only a minimal inhibitory effect on IL-4-mediated proliferation of anti-IgM-activated B cells, IL-4-induced soluble CD23 shedding from both PBMC and T cells depleted of PBMC, and IgE secretion from PBMC, were significantly reduced in a concentration-dependent manner when soluble CD40 was present in the culture. The data presented demonstrate that both soluble forms of the CD40 molecule are biologically active, and suggest that the ligand for CD40 is inducible in IL-4-stimulated cultures and that it mediates both shedding of sCD23 and IgE secretion.

Antibodies, Monoclonal↗

PubMed1992

Molecular and biological characterization of a murine ligand for CD40.

The CD40 surface molecule is a 277-amino-acid glycoprotein expressed on B lymphocytes, epithelial cells and some carcinoma cell lines. Monoclonal antibodies against CD40 mediate a variety of effects on B lymphocytes, including induction of intercellular adhesion, short- and long-term proliferation, differentiation and enhanced tyrosine phosphorylation of proteins. In addition, germinal centre centrocytes are prevented from undergoing apoptosis by activation through CD40 and receptor for antigen. These data indicate that CD40 could be a receptor for an unknown ligand with important functions in B-cell development and activation. This hypothesis is strengthened by the homology of the extracellular region of the CD40 molecule with a family of cell-surface glycoproteins that includes the receptors for nerve growth factor and tumour necrosis factor. Here we report the cloning of a ligand for CD40 that is expressed on the cell surface of activated T cells and mediates B-cell proliferation in the absence of co-stimulus, as well as IgE production in the presence of interleukin-4.

Amino Acid Sequence↗

PubMed1992

Identification of a novel low-affinity receptor for human interleukin-7.

Human recombinant interleukin-7 (IL-7) was labeled with biotin and used to examine IL-7 receptor (IL-7R) expression and regulation on human primary hematopoietic cells, the monocytoid line THP1, and a range of B- and pre-B-celi lines by flow cytometry. A strong intensity of staining was observed using relatively high (greater than 1 x 10(-7) mol/L) concentrations of biotinylated IL-7 on the majority of cell types examined. This reactivity, which could be effectively competed with excess unlabeled IL-7, did not correlate with either mRNA levels for the cloned receptor or with estimates of IL-7R expression determined by [125I]IL-7 binding. Staining of cells with a titration of biotinylated IL-7 showed, at concentrations greater than 1 x 10(-7) mol/L binding with a Ka in the range of 1 x 10(6) mol/L-1, to 1 x 10(7) mol/L-1, an affinity 100 to 1,000 times lower than that reported for the cloned IL-7 receptor. Further data suggesting the existence of a distinct low- affinity IL-7R were provided by two antibodies specific for the cloned IL-7R. Staining with these monoclonal antibodies (MoAbs) correlated with both IL-7R mRNA levels and receptor expression determined by [125I]IL-7 binding, but was not compatible with the distribution of reactivity seen with biotinylated IL-7. Using tritiated biotin to label IL-7, it was estimated that the total number of IL-7 binding sites on the cell lines examined ranged from 1 x 10(4) to at least 5 x 10(5)/cell. Cross-linking studies showed that [125I]IL-7 associated with two major proteins of approximately 62 Kd and 70 Kd on the surface of RPMI 1788 and THP1 cells, in contrast to the 75- to 80 Kd molecule characteristic of the previously cloned receptor, expressed on the surface of Daudi cells. Proliferation of THP1 cells, expressing only the low-affinity form of IL-7R and lacking detectable IL-7R mRNA, could be inhibited by the addition of IL-7 in a concentration-dependent fashion, indicating that, at least on this cell line, binding of IL-7 with a Ka of 1 x 10(6) mol/L-1 to 1 x 10(7) mol/L-1 can transduce a biological signal. Taken together, the data contained in this report demonstrate the existence of a low-affinity IL-7R, expressed in high numbers on hematopoietic cells of different lineages, which is the product of a gene distinct from that encoding the cloned IL-7R.

Biotin↗

PubMed1992

Identification of a source of biologically active CD40 ligand.

We have identified the murine thymoma line EL4 as a source of biologically active CD40 ligand. Using a biotin-labeled soluble CD40.Fc fusion protein, consisting of the extracellular domain of human CD40 and the Fc region of human IgG1, EL4 cells were subjected to repeated flow cytometric cell sorting to select for cells with enhanced biotinylated CD40.Fc binding. After nine rounds of sorting, the number of CD40.Fc binding sites/cell had risen from 450 on the unsorted parental EL4 cells to 15,000 on EL40.9 cells (EL4 cells sorted with biotinylated CD40.Fc for nine rounds). Scatchard analysis of radiolabeled CD40.Fc binding revealed that the surface-expressed CD40 ligand on parental EL4 and EL40.9 cells bound its receptor with a single class of high-affinity sites (Kd = 0.5 nM). Supernatant (SN) from the sorted EL40.9 cells was found to contain human and murine B cell stimulatory activity which could be removed by preclearing with immobilized CD40.Fc, confirming the presence of soluble CD40 ligand in the preparations. EL40.9 supernatant enhanced soluble CD23 (sCD23) release and induced IgE secretion from interleukin 4-stimulated human B cells. In addition, EL40.9 SN contained proliferative activity for anti-IgM-activated murine B cells which could be removed by treatment with immobilized CD40.Fc. However, the same SN had no demonstrable activity on the proliferation of human B cells. The results presented here describe, for the first time, a source of membrane-bound and soluble CD40 ligand. The soluble form of this murine ligand has activity on murine and human B cells and induces some of the functional responses predicted for the ligand based on the action of stimulatory antibodies directed against the human CD40 surface molecule.

Animals↗

PubMed1992

Multiple cytokine secretion by IL-7-stimulated human T cells.

The induction of cytokine secretion by human peripheral blood (PB) T cells was examined. Highly purified T cells stimulated with interleukin 7 (IL-7), in the absence of co-mitogen, secreted IL-2, IL-4, IL-6 and interferon gamma (IFN-gamma) upon restimulation with phorbol ester and ionomycin. In contrast, induction of T-cell cultures initiated with IL-2 or IL-4 yielded only low levels of IL-6 and virtually undetectable levels of IL-4 or IFN-gamma, while IL-2 secretion was reduced. No difference was seen in the ability of CD4+ and CD8+ subpopulations, grown in IL-7, to produce cytokines. In contrast, subdivision of T cells into memory and naive populations using the CD45RO monoclonal antibody (mAb) UCHL1, revealed that almost all of the potential to secrete IL-4 and IL-6 in response to IL-7 resided in the CD45RO+ memory population. Stimulation of cytokine-secreting cells appeared to be a direct effect of IL-7 as neutralizing antibodies directed against IL-2 and IL-4 had no effect on the levels of cytokines produced. The differences observed in the ability of IL-2, IL-4, and IL-7 to potentiate cytokine production was supported by measurement of cytokine mRNA levels by PCR. The elevated levels of cytokine secretion seen in cells cultured with IL-7 was not due simply to increased viability in these cultures compared with those containing IL-2 or IL-4, as these populations showed comparable cloning frequencies in phytohemagglutinin (PHA) + IL-2. These results demonstrate that IL-7, in the absence of co-mitogen, is a potent initial stimulus for multiple cytokine production by human T cells upon restimulation.

Cytokines↗

PubMed1992

Molecular characterization of the interleukin-1 receptor (IL-1R) on monocytes and polymorphonuclear cells.

Primary human monocytes and monocytic cells express an interleukin 1 receptor (IL-1R) which is similar in molecular weight and IL-1 binding characteristics to the IL-1R expressed on B lymphocytes (type II). Northern blot analysis of monocytic cells using a cDNA probe from the recently isolated type II IL-1R indicates that this mRNA is detectable by 4 h and accumulates for at least 24 h following treatment with IL-1R inducing drugs. The time course of induction of this mRNA is slower than that of the type I IL-1R mRNA which is also transcribed in monocytic cells but does not appear to be translated. Sequence analysis of a monocyte-derived cDNA corresponding to the type II IL-1R mRNA shows that the monocyte and B-cell mRNAs are identical. Comparison of monocyte IL-1R peptide maps with those of the type II IL-1R suggests that the two surface IL-1R are identical. This was confirmed serologically using a polyclonal antiserum raised against the type II IL-1R. Data are presented which indicate that primary human neutrophils can also be induced to express abundant type II IL-1R.

Blotting, Northern↗

PubMed1991

Human T cell responses to gp63, a surface antigen of Leishmania.

gp63, an abundant and conserved leishmania cell surface protein, has been implicated in the ability of these parasitic protozoa to infect macrophages in vitro and has shown potential as a protective immunogen in mice. However, little is known regarding human immune responses to this glycoprotein Ag. In this study, human T lymphocyte responses to Leishmania amazonensis native gp63 and to recombinant gp63 (rgp63) produced in Escherichia coli were evaluated in individuals with active or cured cutaneous, mucosal or visceral leishmaniasis. Both native and rgp63 elicited strong proliferative responses in all patients tested. In addition, IFN-gamma was produced in response to stimulation with both forms of the protein. T cell lines generated from PBMC by stimulation with native or rgp63 were phenotypically similar, and proliferated and produced IFN-gamma in response to stimulation with both forms of the molecule. These results suggest that gp63 is a strong T cell immunogen and that the recombinant and native forms can elicit the same type of T cell response from infected patients. In order to compare the immunogenic properties of these two forms of gp63, PBMC from naive (uninfected) donors were sensitized in vitro with native or rgp63. T cell lines generated against rgp63 proliferated in response to rgp63, but failed to proliferate in response to native gp63 or to promastigote lysate. Thus, rgp63 was effective in eliciting T cell responses from patients with active or cured leishmania infection, but did not effectively induce T cell responses under the conditions used.

Animals↗

PubMed1991

Induction of macrophage inflammatory protein-1 beta gene expression in human monocytes by lipopolysaccharide and IL-7.

HuMIP-1 beta is a member of a gene family of inflammatory cytokines whose expression is induced by proinflammatory and mitogenic stimuli. The gene was rapidly induced in human peripheral blood monocytes by either LPS or IL-7. However, IL-7 was unable to induce HuMIP-1 beta mRNA in peripheral blood T cells. The induction of HuMIP-1 beta mRNA in monocytes by IL-7 or LPS was inhibited by IL-4. The 5'-regulatory region of the HuMIP-1 beta gene was cloned and sequenced. An analysis of this sequence revealed three consensus-binding sites for the nuclear factor PU.1 and three potential glucocorticoid response elements. However, the HuMIP-1 beta gene appears to be unresponsive to dexamethasone. Also present in the 5'-regulatory region was a LPS-responsive element located within 455 bp 5' to the start of transcription.

Base Sequence↗

PubMed1991

Expression of interleukin-4 receptors on early human B-lineage cells.

Interleukin-4 (IL-4) regulates multiple stages of the antigen-dependent phase of B-cell development. However, its precise role in regulating B lymphopoiesis in bone marrow is not as well defined. We examined whether surface IgM- normal and leukemic human B-cell precursors (BCP) expressed IL-4 receptors using biotinylated IL-4. Constitutive expression of IL-4 receptors was detected on both normal and leukemic BCP. A higher percentage of normal BCP (82% +/- 15%) expressed IL-4 receptors compared with leukemic BCP (44% +/- 8%). Using mean fluorescent intensity as an indicator of receptor level on the IL-4 receptor positive cells, normal (91 +/- 41) and leukemic (44 +/- 37) BCP expressed comparable numbers of receptors. IL-4 induced the expression of CD23 on 30% of the leukemic BCP cases examined. IL-4 induced CD23 on surface IgM+ fetal bone marrow lymphoid cells but not on the surface IgM- normal BCP, despite the presence of detectable receptors on the surface IgM- cells. IL-4 did not stimulate proliferation of normal BCP, nor could it enhance the effect of recombinant IL-7 or low molecular weight B-cell growth factor. However, IL-4 increased the expression of surface IgM and surface Ig kappa on in vitro differentiated pre-B cells. Our collective results identify no role for IL-4 in the proliferation of normal or leukemic BCP, but identify a role in the enhancement of surface Ig expression during pre-B to B-cell differentiation.

B-Lymphocytes↗

PubMed1991

Expression of receptors for interleukin 4 and interleukin 7 on human T cells.

Human recombinant interleukin 4 (IL-4) and interleukin 7 (IL-7) have been modified with biotin-N-hydroxysuccinimide and used to examine the expression of human IL-4 and IL-7 receptors (R) on activated peripheral blood T cells by flow cytometry. Freshly isolated T cells expressed only a low level of IL-4R which remained unchanged when cells were cultured in the absence of stimuli. In the presence of IL-4, IL-7, phytohemagglutinin A (PHA) or immobilized CD3 monoclonal antibody the intensity of biotinylated IL-4 staining increased approximately twofold on the majority of cells. A combination of mitogen with either IL-4 or IL-7 caused a considerable increase in IL-4 receptor expression over that seen in the presence of mitogen alone. IL-2 alone failed to induce IL-4R although it was able to cause a significant increase in receptor expression on T cells co-cultured with PHA or CD3. Freshly isolated T cells expressed high levels of IL-7R, as determined by biotinylated IL-7 binding and flow cytometry, which did not change significantly with culture in medium alone. Stimulation with PHA, Concanavalin A (Con A) or CD3 had little effect on the intensity of staining. In contrast, activation with phorbol ester resulted in a decrease in IL-7R expression. Similarly, in the presence of IL-4 or IL-7, but not IL-2, the intensity of staining with biotinylated IL-7 was lowered. Analysis of purified T-cell populations showed that IL-7R were present, and IL-4R could be induced, on both CD4+ and CD8+ populations. Analysis of IL-4 receptor expression by this flow cytometric technique was supported by results from 125I-labeled IL-4 binding and by Northern blot analysis of mRNA levels. Taken together, the results of these studies show that the use of biotinylated cytokines and flow cytometry provides a very sensitive method with which to study the expression and regulation of cytokine receptors.

Animals↗

PubMed1990

Regulation of human T cell proliferation by IL-7.

The regulation of human T cell proliferation by rIL-7 was investigated. Purified peripheral blood T cells were stimulated to proliferate in a short-term assay by IL-7 in the presence of CD3 mAb or lectin. This stimulation was accompanied by a significant increase in the expression of IL-2R on both CD4+ and CD8+ T cells over that seen with mitogen alone. The proliferation of these cells in the presence of exogenous IL-7 involved both IL-2-dependent and - independent mechanisms as shown by the ability of neutralizing IL-2 antibody to partially inhibit the response. Anti-IL-4 and anti-IL-6 antibodies had no effect on IL-7-induced T cell growth. These results suggest that the costimulatory effect of IL-7 on human T cells is primarily direct, not involving other intermediate T cell growth factors. IL-7 was also found to be mitogenic in a long-term assay in the absence of any costimulus, with the onset of proliferation occurring later than that seen in the presence of mitogen. These results demonstrate that IL-7 provides a potent T cell stimulus either alone or in the presence of co-mitogen and, although this stimulus is accompanied by an increase in the level of IL-2R expression, it is not dependent on the action of IL-2 for its effect.

Cells, Cultured↗

PubMed1990

A soluble form of the interleukin 4 receptor in biological fluids.

Murine biological fluids and murine cell culture supernatants were analyzed for the presence of soluble murine interleukin 4 receptor (sIL4R) with the use of two monoclonal antibodies directed against the receptor. Mouse urine, serum, ascitic fluid, and cell culture supernatants contained varying levels of immunoreactive protein. All of the immunoreactive protein possessed interleukin 4 (IL 4) binding activity. Following partial purification of ascitic fluid a protein was isolated that binds IL 4 with high affinity. This data is consistent with the fact that murine biological fluids contain a soluble version of the murine IL 4 receptor that arises via secretion of the soluble receptor and/or via shedding of the extracellular portion of the full-length receptor from the cell surface.

Animals↗

PubMed1990

Regulation of interleukin 4 receptors on human T cells.

Human recombinant interleukin 4 (IL-4) was modified with biotin-N-hydroxysuccinimide and used to examine the expression of human IL-4 receptors (IL-4R) on activated peripheral blood T cells by flow cytometry. Freshly isolated T cells expressed only a low level of IL-4R which remained unchanged when cells were cultured in the absence of stimuli. In the presence of IL-4, IL-7, phytohemagglutinin A (PHA), or immobilized CD3 mAb the intensity of biotinylated IL-4 staining increased approximately 2-fold on the majority of cells. A combination of mitogen with either IL-4 or IL-7 caused an increase in receptor expression over that seen in the presence of mitogen alone. IL-2 alone failed to induce IL-4R, although it was able to induce a significant increase in receptor expression on T cells co-cultured with PHA or CD3 mAb. Flow cytometric analysis of purified T cell subsets confirmed that the up-regulation of IL-4R occurred on both CD4+ and CD8+ subpopulations. Two-color staining of T cells activated with PHA and IL-7 revealed that this increase in IL-4R expression occurred almost exclusively on cells expressing the p55 IL-2Ra subunit, although a significant number of cells expressing p55 do not express IL-4R. Analysis of IL-4R expression by this flow cytometric technique was substantiated by 125I-labeled IL-4 binding data and Northern blot analysis of IL-4R mRNA levels, suggesting that use of biotinylated human IL-4 for ligand binding and its detection by flow cytometry provides a very sensitive method for the study of IL-4R regulation.

Biotin↗

PubMed1989

Expression and functional role of CD23 on T cells.

We have found that approximately 10%-15% of tonsil, but not peripheral blood, T cells express the CD23 antigen following activation with 12-O-tetradecanoylphorbol 13-acetate (TPA), phytohemagglutinin (PHA) or recombinant interleukin 4. The proliferative response of tonsil T cells is significantly increased when CD23 monoclonal antibodies (mAb) are present in the cultures. In contrast, no such proliferative augmentation is seen when peripheral blood T cells are cultured in this way. Supernatant (SN) of Epstein-Barr Virus-transformed B lymphoblastoid cell lines (EBVLCL), is found to have a similar co-stimulatory effect on the proliferation of tonsil T cells to that seen with CD23 mAb. This effect is greatly diminished by preclearing SN with CD23 mAb. Similarly, SN from a CD23+ L cell transfectant augments the proliferative response of tonsil T cells to both TPA and PHA. The CD23 molecule expressed by TPA-driven T cell blasts appears identical in size to the 45-kDa glycoprotein present on EBVLCL and activated B cells. In contrast, a 42-kDa molecule is observed when CD23 is precipitated from T cells activated with PHA. The results presented here demonstrate that CD23 is expressed on activated tonsil, but not peripheral blood T cells and plays a role, via the binding of CD23 mAb and CD23+ material, present in EBVLCL and CD23+ transfectant SN, in the regulation of T cell proliferation in response to mitogens such as PHA and TPA.

Adjuvants, Immunologic↗

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

A new antigen identified by the monoclonal antibody UCHB 1 delivers a costimulatory signal to a subset of human B cells.

A novel anti-B cell monoclonal antibody UCHB1 is described which detects an antigen present on a selected subpopulation of both normal and leukemic B cells. Approximately 20% of normal tonsil and peripheral blood (PB) B cells are UCHB 1+ and the majority of B cells from all cases of prolymphocytic leukemia (PLL) tested, together with a low, variable percentage of PB B cells from non-Hodgkin's lymphoma patients with centroblastic centrocytic leukemia also express this antigen. Chronic lymphocytic leukemia and hairy cell leukemia B cells, pre-B acute lymphoblastic leukemia and Epstein-Barr virus transformed cell lines all lack UCHB 1 positivity as do all non-B lineage leukocytes and cell lines so far tested. In tonsil sections UCHB 1 staining is almost completely confined to surface IgM+ mantle zone lymphocytes and follicular dendritic cells, but not B cells, within the germinal centers. UCHB 1 can induce a rise in the level of intracellular free calcium ([Ca2+]i) in PLL B cells and low concentrations of monoclonal antibody can result in the entry of these cells into cell cycle in the absence of additional factors. The proliferative effect of UCHB 1 is greatly enhanced in the presence of Staphylococcus aureus Cowan and, to a lesser extent, phorbol ester. A similar costimulatory effect is exerted on a proportion of normal tonsil B cells although here, despite inducing a rise in [Ca2+]i, UCHB 1 alone does not cause cells to proliferate. UCHB 1 may well prove to be a useful antibody both to distinguish between different B cell leukemias and to study the phenotype and function of leukemic and normal B cell subpopulations.

Animals↗