Search PubMed⌕ Search

Biomedical subjects

K H Grabstein

Publications and source records attributed to K H Grabstein.

At least 55 records · Page 3Linked to original sources
PubMed1993

The regulation of T cell-dependent antibody formation in vitro by CD40 ligand and IL-2.

Our study demonstrates the central role for the murine CD40 ligand and IL-2 in contact-dependent T cell help for Ag-specific primary antibody responses in vitro. Helper T cell clones were found to express CD40 ligand after activation with CD3 mAb. Membrane-bound recombinant CD40 ligand expressed on fixed CV1/EBNA cells had similar B cell-activating properties as T cell clones that had been activated and then fixed. These activities include the induction of B cell proliferation, induction of polyclonal secretion of multiple Ig isotypes in a cytokine-dependent manner, and induction of Ag-specific antibody responses by purified B cells. The induction of polyclonal Ig secretion by the recombinant CD40 ligand required IL-4 and IL-5 although optimal Ag-specific antibody formation required IL-2. Finally, soluble CD40.Fc inhibited the induction of Ag-specific antibody responses by fixed, activated Th cell clones. The requirement for both CD40 ligand and IL-2 for induction of Ag-specific antibody responses was mediated, in part, by the induction of B cell IL-2R expression by CD40 ligand. We conclude that the interaction of CD40 on B cells with its ligand on activated T cells is an integral event in the early activation of B cells to grow and differentiate to antibody formation.

Animals↗
PubMed1993

Recombinant soluble murine IL-4 receptor can inhibit or enhance IgE responses in vivo.

This study examines the effects of soluble IL-4R (sIL-4R) administration on IgE production in vivo by using an anti-IgD injection model. Anti-IgD-treated mice were given various doses of sIL-4R or anti-IL-4 mAb over a 3-day period and serum IgE levels were determined by ELISA on day 9. The sIL-4R inhibited IgE production by up to 85%. Anti-IL-4 mAb administration resulted in comparable levels of inhibition at considerably lower doses. The disparity in efficacy between sIL-4R and anti-IL-4 mAb was likely the result of differences in the biodistribution and in vivo half-life of the two IL-4-binding proteins. The specificity of the sIL-4R inhibitory effect was assessed by mixing sIL-4R with various concentrations of IL-4 before injection. Exogenous IL-4 partially overcame the inhibitory effect of high-dose sIL-4R or anti-IL-4 mAb. Unexpectedly, coadministration of suboptimal concentrations of anti-IL-4 mAb or sIL-4R with IL-4 resulted in superinduction of the IgE response. This stimulatory effect was dose dependent for both IL-4 and the IL-4 cognates and was not seen in the absence of exogenous IL-4 over the entire concentration range tested for either sIL-4R or anti-IL mAb. The results indicate that sIL-4R can block IgE secretion by neutralizing endogenous IL-4. Furthermore, sIL-4R can enhance, in a dose-dependent manner, the biologic effects of exogenously administered IL-4, presumably by altering the biodistribution of the cytokine. These findings suggest two alternative applications for cytokine-binding proteins, i.e., 1) as antagonists of biologic activities of endogenously produced cytokines and, 2) as vehicles for cytokine delivery.

Adjuvants, Immunologic↗
PubMed1993

Characterization of responses of normal human T cells to Trypanosoma cruzi antigens.

The stimulation of normal human PBMC by Trypanosoma cruzi Ag was analyzed. PBMC showed significant in vitro proliferation in response to parasite lysate (Tct), with stimulation indices ranging from 10 to 400, peaking at 6 to 7 days. The cells stimulated with Tct produced significant levels of IL-2. To determine which cells proliferated in response to Tct, PBMC were separated into T- and B-enriched cell populations. Purified T cells, but not B cells, proliferated strongly to Tct. The T cell response required APC and was processing dependent. T cell lines generated against Tct proliferated in response to parasite lysate only in the presence of autologous APC and produced IL-2, IL-6, and IFN-gamma but not IL-4 in response to PMA plus ionomycin. Although there were a significant number of CD45Ra+ cells, the majority of the cells in these T cell lines were CD45Ro+. The V beta usage of Tct-responding T cells was heterogeneous, with most V beta genes represented among the responding cells. An immunodominant repeat Ag (TcD) and a ribosomal phosphoprotein (P0) of T. cruzi elicited strong proliferative responses in all subjects tested. These data indicate the presence of T cell-stimulatory Ag in Tct, characterized by nonpreferential usage of the V beta gene families. The strong stimulation of normal human PBMC by Tct may contribute to immunologic alterations seen in T. cruzi infection.

Animals↗
PubMed1993

Molecular characterization of the early activation antigen CD69: a type II membrane glycoprotein related to a family of natural killer cell activation antigens.

CD69 is a disulfide-linked homo-dimer expressed on the surface of activated T cells, B cells, natural killer cells, neutrophils and platelets. Antibody crosslinking of CD69 in the presence of phorbol ester results in cellular activation events including proliferation and the induction of specific genes. Using an expression cloning strategy we have isolated cDNA encoding human CD69 from a CD4+ T cell clone. Transfection of the cDNA clone in CV-1/EBNA cells results in the expression of a covalently linked homodimer. The cDNA insert hybridizes to a 1.7-kb mRNA in phorbol 12-myristate 13-acetate- or phytohemoagglutinin-stimulated human T cells. Using the human clone we have isolated cDNA encoding mouse CD69, which, when expressed in human T cells allowed those cells to respond to anti-mouse CD69 antibodies by secreting interleukin-2 and interferon-gamma. Sequence analysis showed that both mouse and human CD69 are type II membrane glycoproteins related to the NKR-P1 and Ly-49 families of natural killer cell activation molecules.

Amino Acid Sequence↗
PubMed1993

CD40L: a multi-functional ligand.

B cells can be stimulated to proliferate and differentiate in response to cell-contact dependent signals provided by activated, but not resting, T cells. In the human system, antibodies specific for the surface antigen CD40 induce similar B cell responses. The cloning of a ligand for CD40, and the generation of reagents which can block the interaction of this ligand with its receptor, have demonstrated that the major component of the contact-dependent signal leading to B cell activation is CD40 ligand. Studies of individuals lacking functional CD40 ligand have indicated that signaling through CD40 is essential for immunoglobulin (Ig) heavy chain switching and the production of all isotypes other than IgM. In addition to its activities on B cells, CD40 ligand is stimulatory for cells of monocyte and T lineages suggesting a pleiotropic role for CD40 ligand in vivo.

Animals↗
PubMed1992

Immortalization of murine B cells in vitro with oncogene-containing retroviral vectors.

A large panel of oncogene-containing retroviral vectors has been constructed and used to infect activated murine splenic B cells to determine whether particular oncogenes are capable of directly mediating B cell immortalization. Mature B cell lines have been consistently established with some of these retroviral vectors. These B cell lines arose at a low frequency, indicating that more genetic events were required in addition to infection with the retroviral vector for immortalization to occur. All such lines were LPS-dependent and non-tumorigenic. All lines secrete IgG and express surface IgG, but not IgD or IgM. In addition, they are CD11b+ and CD23-. These cells may be derived from the CD5 "lineage" or a related B cell subset and appear to be more susceptible to immortalization than conventional B cells.

Animals↗
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

Cultured human follicular dendritic cells. Growth characteristics and interactions with B lymphocytes.

Minced human tonsils were digested with DNase and collagenase, and lymphoid cell-depleted low density cells were cultured and grown in granulocyte-macrophage-CSF. Large, morphologically homogenous adherent cells with elongated extensions grew continuously in culture. These nonphagocytic cells appear to be related to follicular dendritic cell (FDC) as they do not have properties of monocytic lineage cells or dendritic cells and because, like FDC, 1) they express CD11b, CD14, CD29, CD40, CD54, CD73, CD74, and VCAM-1, and do not express CD11c, CD22, T cell markers, CD18, CD25 and CD45; and 2) they bind human B lymphocytes and B cell lines, but not T lymphocytes by an adhesion blocked in part by mAb to VLA-4 (CD49d). The cultured FDC also augmented B cell proliferation stimulated by anti-mu sera and/or CD40 mAb. Cultured FDC spontaneously produced low levels of IL-6, but did not produce IL-1 alpha or TNF-alpha; however, after treatment with either IFN-gamma or LPS, they produced more IL-6. The expression of CD54 (ICAM-1) was elevated by treating the cultured FDC with either TNF-alpha, IL-1 beta, IFN-gamma or granulocyte-macrophage-CSF; in contrast, IL-4 had no effect on CD54 but rather up-regulated expression of VCAM-1. IFN-gamma, unlike the other cytokines tested, increased expression of a set of markers on cultured FDC (CD54, VCAM-1, and CD14) and converted these class II-negative cells into class II+ cells. The fact that various T cell-derived cytokines have different effects on FDC suggests that the T cell products may influence the manner by which FDC stimulate B cell proliferation and maturation.

Antigens, CD↗
PubMed1992

Interleukin 10 and interferon gamma regulation of experimental Trypanosoma cruzi infection.

Studies were undertaken to determine whether interleukin 10, (IL-10) a cytokine shown to inhibit interferon gamma (IFN-gamma) production, was involved in Trypanosoma cruzi infections in mice. Exogenous IFN-gamma protects mice from fatal infection with T. cruzi. Furthermore, resistant B6D2 mice developed fatal T. cruzi infections when treated with neutralizing anti-IFN-gamma monoclonal antibody (mAb). Thus, endogenous as well as exogenous IFN-gamma is important in mediating resistance to this parasite. Because both T. cruzi-susceptible (B6) and -resistant (B6D2) mouse strains produced IFN-gamma during acute infection, we looked for the concomitant production of mediators that could interfere with IFN-gamma-mediated resistance to T. cruzi. We found that IL-10-specific mRNA was produced in the spleens of mice with acute T. cruzi infections. In addition, spleen cell culture supernatants from infected B6 mice, and to a lesser extent B6D2 mice, elaborated an inhibitor(s) of IFN-gamma production. This inhibitor(s) was neutralized by anti-IL-10 mAb. These experiments demonstrated the production of biologically active IL-10 during T. cruzi infection. In further studies in vitro, it was shown that IL-10 blocked the ability of IFN-gamma to inhibit the intracellular replication of T. cruzi in mouse peritoneal macrophages. Thus, in addition to its known ability to inhibit the production of IFN-gamma, IL-10 (cytokine synthesis inhibitory factor), may also inhibit the effects of IFN-gamma. These experiments demonstrate that IL-10 is produced during infection with a protozoan parasite and suggest a regulatory role for this cytokine in the mediation of susceptibility to acute disease.

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

Interleukin-3 induces antimicrobial activity against Leishmania amazonensis and Trypanosoma cruzi and tumoricidal activity in human peripheral blood-derived macrophages.

The ability of interleukin-3 (IL-3) to induce antimicrobial and tumoricidal activity was evaluated. Macrophages infected with two intracellular protozoa, Leishmania amazonensis or Trypanosoma cruzi, were treated with cytokines. IL-3 induced a dose-dependent enhancement of microbistasis against leishmanias, and the activity of IL-3 (100 ng/ml) was comparable to that of gamma interferon (IFN-gamma) (1,000 U/ml). In addition, IL-3 in combination with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage CSF (M-CSF) or with IFN-gamma reduced infection and lowered the required dose. IL-3 similarly activated macrophages to inhibit intracellular replication of T. cruzi. Furthermore, IL-3 induced antibody-independent tumoricidal activity against melanoma cells that was dose dependent and comparable to that of lipopolysaccharide and GM-CSF. The mechanisms by which IL-3 induced antimicrobial activity may involve at least the augmentation of oxidative capacity. IL-3, at concentrations of 0.5 ng/ml or greater, led to a significantly increased oxidative burst which paralleled the inhibition of protozoan replication. The enhancement of oxidative capacity by IL-3 (5 ng/ml or higher) was comparable to that of IFN-gamma. The induction of tumoricidal activity was associated with the production of tumor necrosis factor alpha (TNF-alpha), which in this system may feed back to enhance the macrophage inhibition of leishmanias, as demonstrated by neutralization of IL-3 activation by anti-TNF-alpha antibody. Thus, peripheral blood macrophages remain responsive to IL-3, as demonstrated by enhanced antimicrobial and tumoricidal activity. IL-3 may have potential clinical applications because of these properties and its effect on myelopoiesis.

Animals↗
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

Efficient gene transfer and expression in primary B lymphocytes.

In this study we have used a panel of vectors expressing the chloramphenicol acetyltransferase (CAT) reporter gene under the control of different regulatory elements to optimize gene transfer and expression in primary B lymphocytes. The Moloney murine leukemia virus long terminal repeat (MoMLV LTR) and the SV40 early region promoters, while functional in transfected plasmacytoma cell lines, did not give rise to detectable CAT activity following transfection into primary activated mouse or human B lymphocytes. In contrast, the human cytomegalovirus immediate-early (HCMV-IE) enhancer/promoter functioned in both established and primary B cells. The highest expression levels in the primary cells were obtained with vectors containing the Adenovirus 2 major late promoter or the HCMV-IE enhancer/promoter in combination with the Adenovirus 2 tripartite leader and VA genes. These latter expression cassettes were placed in a retroviral vector with the aim of combining their capacity for high-level gene expression with the efficient stable gene transfer afforded by retroviral infection. Several retroviral constructs were made, some of which were able to generate high virus titers. However all of these underwent deletions during the process of retroviral infection, as judged by Southern analysis of infected cells, indicating that they were not optimal gene transfer vectors. The HCMV enhancer/promoter, which was the most active of the other expression cassettes tested in the primary B cells, was inserted into a retroviral vector which also expressed the hph gene under the transcriptional control of the retroviral LTR. This vector did not undergo rearrangement during the process of retroviral infection, as judged by Southern analysis. The CAT gene was inserted downstream of the HCMV promoter in this vector, and a high-titer retroviral stock was generated. Primary B lymphocytes infected with this vector gave high levels of CAT activity, under conditions in which parallel experiments with the hph drug resistance marker showed that one in 20 of the cells were infected. These experiments demonstrate efficient gene transfer and expression in primary B lymphocytes in vitro.

Animals↗
PubMed1991

Interleukin 7 induces cytokine secretion and tumoricidal activity by human peripheral blood monocytes.

Peripheral blood monocytes can be induced by stimuli such as bacterial lipopolysaccharide (LPS) to secrete an array of cytokines. We have studied the effects of interleukin 7 (IL-7) on human peripheral blood mononuclear cells (PBMC) and found that IL-7 is a relatively potent inducer of IL-6 secretion IL-6 protein levels were determined either by the B9 hybridoma growth factor assay or by enzyme-linked immunosorbent assay, and mRNA for IL-6 was analyzed by Northern hybridization. Detailed examination revealed that, among PBMC, monocytes, rather than lymphocytes, were secreting IL-6 in response to IL-7. In contrast to the low concentrations of IL-7 required to stimulate T cell growth and differentiation (as low as 0.1 ng/ml), relatively high concentrations of IL-7 were necessary to induce IL-6 secretion by monocytes (at least 10 ng/ml). An optimal concentration of IL-7 (100 ng/ml) induced monocytes to secrete 10-fold more IL-6 than an optimal concentration of IL-1 beta (10 ng/ml), and almost as much as LPS. However, significantly more IL-7 than IL-1 beta was required to induce detectable levels of IL-6. The kinetics of IL-6 secretion by monocytes were identical in response to IL-7, IL-1 beta, or LPS, with IL-6 protein detectable in culture supernatants as early as 2 h after the initiation of culture. IL-4 was found to markedly inhibit the ability of IL-7 or LPS to induce IL-6 mRNA and IL-6 secretion. In addition to promoting IL-6 production, IL-7 induced the secretion of immunoreactive IL-1 alpha, IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) by monocytes. IL-7 also induced monocyte/macrophage tumoricidal activity against a human melanoma cell target, an activity that may be related to the secretion of IL-1 alpha, IL-1 beta, and TNF-alpha. Finally, we used a whole blood culture system as a bridge to in vivo analysis to demonstrate that IL-7 induces cytokine secretion in the absence of culture medium, fetal calf serum, and adherence to plastic. Our data suggest that IL-7, in addition to regulating lymphocyte growth and differentiation, has potent effects on cells of the monocytic lineage. Thus, IL-7 may be an important mediator in inflammation and in the macrophage immune response to tumors.

Cell Survival↗
PubMed1990

Recombinant granulocyte-macrophage colony-stimulating factor restores deficient immune responses in mice with chronic Trypanosoma cruzi infections.

Spleen cells from mice with chronic Trypanosoma cruzi infection generate a minimal plaque-forming response to SRBC in vitro. Addition of granulocyte-macrophage (GM)-CSF to cultures of spleen cells from chronically infected mice restored the plaque-forming cells (PFC) response to normal levels. Splenic adherent cells from chronically infected mice were deficient in their ability to reconstitute the PFC response of accessory cell-depleted normal spleen cells. Preincubation of splenic adherent cells from infected mice with GM-CSF restored their ability to reconstitute the PFC response of adherent cell depleted cultures. Ia Ag expression by splenic adherent cells from chronically infected mice was significantly lower compared to Ia Ag expression of cells from normal mice. Incubation of splenic adherent cells from chronically infected mice for 48 h with GM-CSF increased levels of Ia Ag expression to approximately those of uninfected mice. Peritoneal macrophages from infected mice produced IL-1 after incubation with GM-CSF at levels equivalent to those produced by similarly treated control macrophages. Spleen cells from chronically infected mice showed significant induction of IL-2 mRNA after GM-CSF treatment, and the addition of the anti-IL-2 mAb to GM-CSF supplemented cultures of spleen cells from infected mice blocked the restoration of the anti-SRBC PFC response. Thus, the ability of GM-CSF to restore the anti-PFC response to SRBC appears to involve the up-regulation of accessory cell function that includes increased Ia Ag expression and the induction of IL-1 production. These events also involve increased IL-2 production with resultant up-regulation of the response to SRBC by spleen cells from infected mice. Finally, it was shown that treatment of infected mice with rGM-CSF completely restored their depressed PFC production in vivo.

Animals↗
PubMed1990

T-cell growth factor P40 promotes the proliferation of myeloid cell lines and enhances erythroid burst formation by normal murine bone marrow cells in vitro.

T-cell growth factor P40 was examined for possible effects on murine interleukin-3 (IL-3)-dependent myeloid cell lines and freshly isolated murine bone marrow cells. The results showed that P40 stimulated the proliferation of some IL-3-dependent myeloid cell lines of both early myeloid and mast cell phenotype and synergized with IL-3. P40 did not promote proliferation of fresh bone marrow cells, bone marrow enriched for early myeloid cells by 5-fluorouracil treatment, or bone marrow derived mast cells as assessed in 3H-TdR incorporation assays. P40 did not influence the growth of murine colony-forming unit granulocyte-macrophage in agar cultures, either alone or in the presence of optimal or sub-optimal concentrations of CSF-1, GM-colony-stimulating factor, or IL-3. P40 did potentiate burst-forming unit-erythroid (BFU-E) formation in the presence of erythropoietin; however, this was dependent on the cell plating density, suggesting an indirect stimulation of BFU-E by P40. The indirect nature of P40 action on BFU-E was further demonstrated in cell separation experiments and indicated that the effect was mediated by T cells. These data expand the repertoire of cells that P40 influences.

Animals↗
PubMed1990

Regulation of T cell proliferation by IL-7.

The regulation of murine T cell proliferation by IL-7 was investigated. Highly purified resting splenic T cells were induced to proliferate in a short term assay by IL-7 in the presence of the comitogen, Con A. The proliferation of these resting T cells showed both IL-2-dependent and -independent components as determined by the susceptibility of the response to the blocking effects of anti-IL-2 mAb. Furthermore, IL-7 was found to augment the Con A-induced production of IL-2 and expression of IL-2R by resting splenic T cells. In contrast, Con A blasts and long term, Ag-dependent cloned T cells proliferated in response to IL-7 independently of any involvement of IL-2. Finally, differences were observed between IL-7 and IL-6 with regard to the regulation of T cell growth and activation. As with IL-7, IL-6 stimulated resting splenic T cells to proliferate in the presence of comitogen. However, in contrast to IL-7, IL-6 failed to stimulate the proliferation of Con A blasts or T cell clones and did not augment the Con A-induced expression of IL-2R on resting T cells.

Animals↗