Search PubMed⌕ Search

Biomedical subjects

C H June

Publications and source records attributed to C H June.

At least 127 records · Page 7Linked to original sources
PubMed1991

Variations in thymocyte susceptibility to clonal deletion during ontogeny. Implications for neonatal tolerance.

Activation of immature thymocytes via the TCR results in programmed cell death and clonal deletion. We have examined thymocytes from mice of different ages and observed that, whereas TCR-mediated signaling caused deletion of thymocytes from newborn and 3-week-old mice, it failed to delete thymocytes from mice of 1 week of age. This could not be attributed to differences in cell surface TCR expression, TCR-mediated phosphoinositide hydrolysis or Ca2+ mobilization, or total cellular levels of TCR zeta- and eta-chains. Moreover, thymocytes of all ages were equally susceptible to corticosteroid- and Ca2+ ionophore-induced programmed cell death. These data are consistent with the notion that fetal and neonatal thymocytes represent a relatively synchronous wave of cells passing through phases in which they are susceptible and then resistant to TCR-induced programmed cell death. They also support the notion that the classical phenomenon of neonatal tolerance is due to clonal deletion and that the inability of allogeneic cells to tolerize mice at 1 week of age is because the thymocytes are refractory to TCR-alpha beta-mediated clonal deletion.

Age Factors↗
PubMed1991

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Signal transduction in T cells.

Rapid progress was made during the past year in the delineation of the nature of the initial biochemical events triggered by the T-cell antigen receptor. Antigen-mediated activation of phospholipase C was demonstrated to require protein tyrosine phosphorylation and, most surprising, activation of the Ras family of signal transduction molecules was shown to closely follow stimulation of the T-cell antigen receptor. Major controversy continues over which events are relevant to the various effector functions of T cells.

Animals↗
PubMed1991

An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3' untranslated region of lymphokine mRNA.

Considerable evidence suggests that the metabolism of lymphokine mRNAs can be selectively regulated within the cytoplasm. However, little is known about the mechanism(s) that cells use to discriminate lymphokine mRNAs from other mRNAs within the cytoplasm. In this study we report a sequence-specific cytoplasmic factor (AU-B) that binds specifically to AUUUA multimers present in the 3' untranslated region of lymphokine mRNAs. AU-B does not bind to monomeric AUUUA motifs nor to other AU-rich sequences present in the 3' untranslated region of c-myc mRNA. AU-B RNA-binding activity is not present in quiescent T cells but is rapidly induced by stimulation of the T-cell receptor/CD3 complex. Induction of AU-B RNA-binding activity requires new RNA and protein synthesis. Stabilization of lymphokine mRNA induced by costimulation with phorbol myristate acetate correlates inversely with binding by AU-B. Together, these data suggest that AU-B is a cytoplasmic regulator of lymphokine mRNA metabolism.

Base Sequence↗
PubMed1991

UV irradiation of lymphocytes triggers an increase in intracellular Ca2+ and prevents lectin-stimulated Ca2+ mobilization: evidence for UV- and nifedipine-sensitive Ca2+ channels.

UV irradiation induces in vitro and in vivo immunosuppression. Because mobilization of intracellular calcium ([Ca2+]i) represents a central step in cell activation and immune response, we investigated the effect of UV irradiation on Ca2+ homeostasis. Using indo-1 and cytofluorometry, [Ca2+]i kinetics in UVC- or UVB-exposed human peripheral blood leukocytes (PBL) and Jurkat cells were determined in parallel with functional assays. Increases in [Ca2+]i were observed within 2-3 h of irradiation; these increases were UV-dose dependent and reached maxima of 240% and 180% above baseline level (130 nM) for UVB and UVC, respectively. The UV-induced [Ca2+]i rise was predominantly due to influx of extracellular calcium, and it was more pronounced in T than in non-T cells. Concurrent with [Ca2+]i shifts following UV treatment, there was a loss of ability to respond to phytohemagglutinin (PHA) or to proliferate or stimulate in mixed leukocyte culture. This loss of function appeared to be related not only to UV-induced calcium shifts, but also to effects of UV irradiation on the plasma membrane. No [Ca2+]i mobilization was induced by gamma irradiation, and gamma-irradiated cells showed a normal [Ca2+]i increase in response to PHA. UV-induced Ca2+ flux into the cells was blocked by nifedipine. These data indicate that UV and gamma irradiation have different effects on lymphocyte membranes and suggest that a disruption of Ca2+ homeostasis may be involved in UV-induced lymphocyte inhibition. The data suggest, furthermore, the presence of Ca2+ channels in lymphocyte membranes that are sensitive to UV irradiation and Ca2+ channel blockers such as nifedipine.

Calcium↗
PubMed1990

No T-cell tyrosine protein kinase signalling or calcium mobilization after CD4 association with HIV-1 or HIV-1 gp120.

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.

Antibodies, Monoclonal↗
PubMed1990

IL-5 induces a Pgp-1 (CD44) bright B cell subpopulation that is highly enriched in proliferative and Ig secretory activity and binds to hyaluronate.

Pgp-1 expression was examined in unstimulated B cell populations and in B cells activated with several polyclonal stimuli. Flow cytometry analysis demonstrated that Pgp-1 expression increased when B cells were activated with supernatant of cloned Th2 cells, with LPS, or with IL-5, stimuli that induced polyclonal proliferation and differentiation. IL-5-primed B cells were phenotypically unique and could be divided into two distinct subpopulations based on the brightness of Pgp-1 expression. Furthermore, sterile sorting experiments showed that proliferating and differentiating B cells were highly enriched in a Pgp-1-bright, Ia-dull, B220-dull subpopulation. The possibility that Pgp-1 expressed on activated B cells functions as an adhesion molecule was evaluated by assessing adhesion of activated B cells to defined substrates. It was found that IL-5-activated B cells bound strongly to hyaluronate-coated surface, and this binding was specifically inhibited by anti-Pgp-1 Ab. These findings suggest that Pgp-1 expression is a useful marker which, under defined conditions, identifies the proliferating and differentiating subset of activated B cells. Moreover, the Pgp-1 bright subset of IL-5-primed B cells binds to hyaluronate in a Pgp-1-dependent manner that suggests a potential role of Pgp-1 in the in vivo adherence and trafficking of activated B cells.

Animals↗
PubMed1990

Activation of tyrosine phosphorylation in human T cells via the CD2 pathway. Regulation by the CD45 tyrosine phosphatase.

In this study we compare the effect of CD3 and CD2 ligation on tyrosine kinase activation in human peripheral blood T cells. Using antiphosphotyrosine antibody to detect tyrosine phosphorylation of cellular substrates, we demonstrate that mAb stimulation of either CD3 or CD2 results in tyrosine phosphorylation of the TCR-zeta chain and 135- and 100-kDa proteins. However, differences are observed between CD3 and CD2 ligation; only the former results in rapid tyrosine phosphorylation of 72-, 65-, and 40-kDa substrates. Co-aggregation of CD2 and CD45, a tyrosine phosphatase, results in inhibition of intracellular calcium elevation and T cell proliferation. We demonstrate in this study that this manipulation also inhibits polyphosphoinositide hydrolysis and tyrosine phosphorylation of the 100-kDa substrate. The failure of tyrosine phosphorylation of the 100-kDa substrate is specific in that phosphorylation of the 135-kDa protein is not inhibited. Similar results are observed when CD2 and CD45 are independently cross-linked rather than co-aggregated. The observation that CD45 cross-linking alters tyrosine phosphorylation of T cell substrates and effects polyphosphoinositide hydrolysis is further evidence that tyrosine phosphorylation regulates early events in T cell activation including, perhaps, phospholipase C activity.

Antigens, CD↗
PubMed1990

Inhibition of T cell receptor expression and function in immature CD4+CD8+ cells by CD4.

Most immature CD4+CD8+ thymocytes express only a small number of T cell receptor (TCR) molecules on their surface, and the TCR molecules they do express are only marginally capable of transducing intracellular signals. TCR expression and function was not intrinsically low in immature CD4+CD8+ thymocytes, but was found to be actively inhibited by CD4-mediated signals. Indeed, release of CD4+CD8+ thymocytes from CD4-mediated signals resulted in significant increases in both TCR expression and signaling function. These results suggest that, in CD4+CD8+ cells developing in the thymus, increased TCR expression and function requires release from CD4-mediated inhibition.

Animals↗
PubMed1990

The genomic organization of the CD28 gene. Implications for the regulation of CD28 mRNA expression and heterogeneity.

CD28 is a 90-kDa homodimeric glycoprotein present on the surface of a large subset of T cells that appears to play an important role in the modulation of T cell activation. Although a number of physiologic effects associated with CD28 stimulation have been defined, relatively less is known about the structure and expression of the CD28 gene itself. We now show that CD28 is expressed in both Th cells and plasma cells as a series of four distinct CD28 mRNA species: 1.3-, 1.5-, 3.5-, and 3.7-kb transcripts. The steady state expression of all four transcripts in CD28+ T cells was stimulated by PMA, suggesting that they might share a common phorbol-sensitive promoter. Consistent with this hypothesis, CD28 was found to be encoded by a single copy gene organized into four exons, each exon defining a functional domain of the predicted protein. All CD28 transcripts appear to initiate within a 61-bp palindrome. Generation of the four CD28 mRNA species from the CD28 gene involves two distinct posttranscriptional events. The longer pair of transcripts (3.5/3.7 kb) is generated by the use of an alternate nonconsensus polyadenylation signal. This results in the addition of 2167 bp beyond the first polyadenylation site utilized by the shorter (1.3/1.5 kb) pair of transcripts. The size difference between the 3.7- and 3.5-kb messages and between the 1.5- and 1.3-kb messages is generated by an internal splicing event that deletes 252 bp within exon 2, which encodes the extracellular domain. This deletion would result in the loss of 84 amino acids, including 4 of 5 extracellular cysteine residues. Although this deletion would result in significant disruption of CD28 secondary structure, it would not be expected to interfere with the ability of the resultant protein to be expressed on the cell surface. These findings suggest that variant isotypes of CD28 may be expressed on the cell surface with potentially different physiologic roles.

Amino Acid Sequence↗
PubMed1990

CD28 ligation in T-cell activation: evidence for two signal transduction pathways.

The CD28 homodimer is thought to function as a signal transducing receptor during activation of T cells. Evidence is presented that the degree of aggregation of CD28 on the cell surface regulates two distinct CD28-associated signals. Binding of bivalent CD28 monoclonal antibody (MoAb) 9.3 upregulates lymphokine production by messenger RNA (mRNA) stabilization, without direct initiation of lymphokine mRNA transcription. This signal was not dependent on inositol phospholipid production or activation of a protein tyrosine kinase (PTK). In contrast, further crosslinking of CD28 on the cell surface rapidly induced formation of large amounts of inositol trisphosphate (InsP3) and increased cytoplasmic calcium concentration [( Ca2+]i), but did not stimulate PTK. CD28 crosslinking directly activated a subset of resting T cells, since CD25 (interleukin [IL]-2 receptor alpha chain) mRNA was rapidly induced in purified T cells, and proliferation, even without addition of exogenous IL-2, was sometimes observed. CD25 expression was detected on the cell surface of approximately 20% of CD4+ T cells. The degree of CD28 aggregation required for activation was investigated by preparing soluble 9.3 x 9.3 conjugates ranging in size from approximately 300 Kd to greater than 1,000 Kd, and comparing their function in T-cell proliferation assays with phorbol-12-myristate-13-acetate (PMA), anti-CD3, or IL-2. There was a correlation between conjugate size and proliferation with IL-2, whereas costimulation with PMA or CD3 was optimized at a lower degree of CD28 aggregation. The inositol phospholipid (InsP) generation and increase in [Ca2+]i after CD28 receptor aggregation appeared to proceed through a pathway different from the CD3/T-cell receptor (TCR) pathway since it was enhanced by pretreatment with PMA, while the InsP and [Ca2+]i signal from crosslinking CD3 was suppressed by PMA. Furthermore, the proliferation response to CD28 aggregation was resistant to inhibition by CD3 modulation. Thus, CD28 aggregation appears to trigger a phospholipase C activation pathway that differs from the CD3/TCR-linked pathway.

Animals↗
PubMed1990

Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation.

Antiphosphotyrosine immunoblots were used to characterize tyrosine phosphorylated proteins after stimulation of the human TCR. Increased tyrosine phosphorylation was evident on at least 12 substrates within 2 min after ligation of the TCR with mAb. Analysis of the time course for increased tyrosine phosphorylation revealed distinct patterns. Increased phosphorylation of 135-kDa and 100-kDa substrates was evident within 5 s, whereas increased phosphorylation of the TCR-zeta-chain required several minutes after treatment with anti-CD3 mAb. This rapid cellular tyrosine phosphorylation occurred independent of the cell cycle, as it occurred after stimulation of resting T cells, T cell blasts, and the Jurkat T cell leukemia line. When the TCR complex was cross-linked together with the CD4 receptor by heteroconjugate anti-CD3/CD4 mAb, an increased magnitude of tyrosine phosphorylation occurred, although no new substrates could be detected. The increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates was specific in that anti-HLA class I, anti-CD6, anti-CD7, and anti-CD28 antibodies did not cause increased tyrosine phosphorylation. Anti-CD4 stimulation of resting T cells did not cause increased tyrosine phosphorylation of pp100 and pp135, suggesting that the CD4-associated kinase, lck, does not account for the tyrosine phosphorylation observed after TCR stimulation. Similarly, pharmacologic treatment of cells with phorbol ester and calcium ionophore did not cause increased tyrosine phosphorylation of these substrates, indicating that activation of protein kinase C or phospholipase C does not account for these early increases in tyrosine phosphorylation. The time of onset of pp100 phosphorylation, and the magnitude of phosphorylation correlated with the magnitude of calcium mobilization when cells were stimulated with different forms of TCR stimulation. When cells were labeled with [3H]myoinositol and analyzed after stimulation by anti-CD3 mAb, increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates preceded the activation of phospholipase C, as measured by the appearance of inositol 1,4,5-trisphosphate. This occurred in both T cell blasts and in the Jurkat T cell line. Thus, these findings show that increased tyrosine phosphorylation is the earliest yet detected signal observed after ligation of the TCR complex, and furthermore suggest that tyrosine phosphorylation might link the TCR to the phosphatidylinositolbisphosphate hydrolysis signaling pathway.

Animals↗
PubMed1990

Monovalent ligands of complement receptor 2 inhibit whereas polyvalent ligands enhance anti-Ig-induced human B cell intracytoplasmic free calcium concentration.

We have performed experiments to investigate the role of ligands for complement receptor 2 (CR2) in human B cell activation. Flow microfluorimetry was used to assess changes in free intracytoplasmic calcium concentration [Ca2+] in indo-loaded B cells, immediately after exposure to anti-mu antibody and to monovalent or polyvalent CR2 ligands. As monovalent ligands we used the C3d fragment and synthetic C3 peptides (peptides P14, residues 1201-1214, and P28, residues 1187-1214). As polyvalent ligands we used i) an intact monoclonal mouse anti-CR2 antibody (HB5) and its F(ab')2 fragment, ii) tetravalent P13 [residues 1202-1214) 4-template), and iii) P28 conjugated to BSA (molar ratio 5/1). Anti-CR2 antibody HB5, tetravalent P13, and P28 conjugated to BSA, enhanced the ability of F(ab')2 fragments of the IgG fraction of goat anti-human mu antibody to increase human B cell [Ca2+]i. In contrast, the monomeric CR2 ligands C3d and P28 inhibited the anti-mu-induced increase in human B cell [Ca2+]i. Multivalent P13, P28, and the HB5, by themselves, did not affect B cell [Ca2+]i. These experiments suggest that the valence of the CR2 ligands is crucial for the nature (synergistic vs antagonistic) of the message transmitted through the CR2.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1990

CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes.

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.

Antibodies, Monoclonal↗
PubMed1990

Protein kinase C activation in B cells by indolactam inhibits anti-Ig-mediated phosphatidylinositol bisphosphate hydrolysis but not B cell proliferation.

In order to examine the role of phosphatidylinositol bisphosphate (PIP2) hydrolysis in B cell activation, we studied the effect of various classes of protein kinase C (PKC) activators on anti-Ig-mediated B cell stimulation. Anti-Ig-stimulated PIP2 hydrolysis, elevations in [Ca2+]i, and induction of DNA synthesis were inhibited by PMA (a phorbol ester) as previously reported. In contrast, indolactam (an alkaloid PKC activator) inhibited PIP2 hydrolysis and elevations in [Ca2+]i, but stimulated rather than inhibited cellular proliferation. In order to examine whether the binding avidity of the PKC activators to PKC played a role in determining their activity to stimulate or inhibit B cell activation, we studied two other PKC activators, bryostatin and mezerein. Again, both inhibited anti-Ig mediated PIP2 hydrolysis and elevations in [Ca2+]i, whereas only the former inhibited induction of DNA synthesis. These data suggest that a) high levels of PIP2 hydrolysis and elevations in [Ca2+]i are not essential for anti-Ig-mediated induction of B cell DNA synthesis and b) activation of PKC may induce both stimulatory and inhibitory pathways of B cell activation, and whether stimulation or inhibition of cell activation is observed may depend on the combined intensity of these two signals.

Animals↗
PubMed1990

Activation of human B lymphocytes by nanogram concentrations of anti-IgM-dextran conjugates.

Surface immunoglobulin (sIg) cross-linking on B lymphocytes by high concentrations of anti-Ig antibody has been used to mimic antigen-stimulated B cell activation. In order to develop a system to study sIg-mediated T cell-independent B cell activation using low concentrations of anti-Ig antibody that more closely resemble the concentrations of antigen that are achieved under in vivo conditions, we conjugated monoclonal anti-human IgM antibody (anti-mu) to dextran (molecular weight 2 X 10(6)) thereby increasing its valency. This dextran conjugate (anti-mu-Dex) stimulated comparable levels of thymidine incorporation and B cell size increases as were seen with unconjugated anti-mu but at 100- to 1000-fold lower concentrations. Anti-mu-Dex also stimulated increases in intracellular ionized calcium ([Ca2+]i) in a higher percentage of cells, of greater magnitude and of longer duration than that stimulated by unconjugated anti-mu. Interestingly, there was no direct correlation between the increases in [Ca2+]i that were stimulated by anti-mu-Dex and its ability to stimulate B cell proliferation. The concentrations of anti-mu-Dex (10 micrograms/ml) that led to the highest increase in [Ca2+]i resulted in thymidine incorporation that was no greater than that of medium control, whereas 0.01 to 0.1 microgram/ml stimulated significant thymidine incorporation with 50% lower levels of stimulation of [Ca2+]i. These data demonstrate that anti-mu-Dex is a potent activator of human B lymphocytes, is effective even at ng/ml concentrations which over a 2-h time period do not induce detectable modulation of sIg, and its stimulation of B cells into G1 and S may not be directly related to its ability to stimulate increases in levels of [Ca2+]i.

Antibodies, Anti-Idiotypic↗
PubMed1990

Role of the CD28 receptor in T-cell activation.

Antigen-specific T-cell activation is initiated through the T-cell receptor. Recent evidence has shown that a number of additional T-cell surface receptors serve to regulate the responses of antigen-activated T cells. One such molecule, CD28, is a member of a heterophilic cell adhesion complex, and is the receptor for the B-cell-restricted B7/BB-1 antigen. As Carl June, Jeffrey Ledbetter, Peter Linsley and Craig Thompson review here, CD28 serves as the surface component of a novel signal transduction pathway that modulates T-cell lymphokine production and increases the resistance of T-cell responses to various immunosuppressive agents.

Animals↗