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M Julius

Publications and source records attributed to M Julius.

At least 19 recordsLinked to original sources
PubMed2001

Role for lipid rafts in regulating interleukin-2 receptor signaling.

Lipid rafts are plasma membrane microdomains characterized by a unique lipid environment enriched in gangliosides and cholesterol, leading to their insolubility in nonionic detergents. Many receptors are constitutively or inducibly localized in lipid rafts, which have been shown to function as platforms coordinating the induction of signaling pathways. In this report, the first evidence is provided for a role of these lipid microdomains in regulating interleukin-2 receptor (IL-2R) signaling. It is demonstrated that antibody- or ligand-mediated immobilization of components of lipid rafts, glycosyl-phosphatidyl-inositol-anchored proteins, and the GM1 ganglioside, respectively, inhibit IL-2-induced proliferation in T cells. IL-2Ralpha is shown to be constitutively enriched in rafts and further enriched in the presence of immobilized anti-Thy-1. In contrast, IL-2Rbeta and IL-2Rgamma, as well as JAK1 and JAK3, are found in soluble membrane fractions, and their localization is not altered by anti-Thy-1. IL-2-mediated heterotrimerization of IL-2R chains is shown to occur within soluble membrane fractions, exclusively, as is the activation of JAK1 and JAK3. As predicted by these results, the disruption of lipid raft integrity did not impair IL-2-induced signaling. Thus, the sequestration of IL-2Ralpha within lipid microdomains restricts its intermolecular interactions and regulates IL-2R signaling through impeding its association with IL-2Rbeta and IL-2Rgamma.

Animals↗
PubMed2001

Soluble CD14 enriched in colostrum and milk induces B cell growth and differentiation.

Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiation in vitro is induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14 in vitro, its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin.

Animals↗
PubMed2001

Synergistic regulation of immunoreceptor signaling by SLP-76-related adaptor Clnk and serine/threonine protein kinase HPK-1.

Recently, the identification of Clnk, a third member of the SLP-76 family of adaptors expressed exclusively in cytokine-stimulated hemopoietic cells, has been reported by us and by others. Like SLP-76 and Blnk, Clnk was shown to act as a positive regulator of immunoreceptor signaling. Interestingly, however, it did not detectably associate with known binding partners of SLP-76, including Vav, Nck, and GADS. In contrast, it became complexed in activated T cells and myeloid cells with an as yet unknown tyrosine-phosphorylated polypeptide of approximately 92 kDa (p92). In order to understand better the function of Clnk, we sought to identify the Clnk-associated p92. Using a yeast two-hybrid screen and cotransfection experiments with Cos-1 cells, evidence was adduced that p92 is HPK-1, a serine/threonine-specific protein kinase expressed in hemopoietic cells. Further studies showed that Clnk and HPK-1 were also associated in hemopoietic cells and that their interaction was augmented by immunoreceptor stimulation. A much weaker association was detected between HPK-1 and SLP-76. Transient transfections in Jurkat T cells revealed that Clnk and HPK-1 cooperated to increase immunoreceptor-mediated activation of the interleukin 2 (IL-2) promoter. Moreover, the ability of Clnk to stimulate IL-2 promoter activity could be blocked by expression of a kinase-defective version of HPK-1. Lastly we found that in spite of the differential ability of Clnk and SLP-76 to bind cellular proteins, Clnk was apt at rescuing immunoreceptor signaling in a Jurkat T-cell variant lacking SLP-76. Taken together, these results show that Clnk physically and functionally interacts with HPK-1 in hemopoietic cells. Moreover, they suggest that Clnk is capable of functionally substituting for SLP-76 in immunoreceptor signaling, albeit by using a distinct set of intracellular effectors.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2.

Glycosylphosphatidylinositols (GPIs) are attached to the C-termini of many proteins, thereby acting as membrane anchors. Biosynthesis of GPI is initiated by GPI-N-acetylglucosaminyltransferase (GPI-GnT), which transfers N-acetylglucosamine from UDP- N-acetylglucosamine to phosphatidylinositol. GPI-GnT is a uniquely complex glycosyltransferase, consisting of at least four proteins, PIG-A, PIG-H, PIG-C and GPI1. Here, we report that GPI-GnT requires another component, termed PIG-P, and that DPM2, which regulates dolichol-phosphate-mannose synthase, also regulates GPI-GnT. PIG-P, a 134-amino acid protein having two hydrophobic domains, associates with PIG-A and GPI1. PIG-P is essential for GPI-GnT since a cell lacking PIG-P is GPI-anchor negative. DPM2, but not two other components of dolichol-phosphate-mannose synthase, associates with GPI-GnT through interactions with PIG-A, PIG-C and GPI1. Lec15 cell, a null mutant of DPM2, synthesizes early GPI intermediates, indicating that DPM2 is not essential for GPI-GnT; however, the enzyme activity is enhanced 3-fold in the presence of DPM2. These results reveal new essential and regulatory components of GPI-GnT and imply co-regulation of GPI-GnT and the dolichol-phosphate-mannose synthase that generates a mannosyl donor for GPI.

Amino Acid Sequence↗
PubMed2000

Intestinal inflammation observed in IL-2R/IL-2 mutant mice is associated with impaired intestinal T lymphopoiesis.

BACKGROUND & AIMS: Although interleukin (IL)-2(-/-) and IL-2Ralpha(-/-) mice develop inflammatory bowel disease, IL-2Rbeta(-/-) animals are apparently free of gut pathology. Intraintestinal T lymphopoiesis is reported to be impaired in IL-2Rbeta(-/-) mice; we have determined whether this characteristic correlated with the apparent resistance of this mutant strain to intestinal inflammation. This led us to reassess intraintestinal T lymphopoiesis in these 3 mutant strains. METHODS: Intestinal histology and intraintestinal T lymphopoiesis were analyzed in unmanipulated mutant mice and in athymic and euthymic radiation chimeras reconstituted with bone marrow derived from IL-2(-/-), IL-2Ralpha(-/-), and IL-2Rbeta(-/-) donors. RESULTS: Intraintestinal T lymphopoiesis was ablated in the 3 mutant strains and was associated with cryptopatch abnormalities. The intestinal mucosa of mice reconstituted with lymphocytes from IL-2Rbeta(-/-) mice exhibited lesions of both the small and large bowel similar to those observed in the early stages of human gluten enteropathy and acute ulcerative colitis, respectively. Analysis of euthymic and athymic bone marrow radiation chimeras indicated that T cells located in the intestinal mucosa of unmanipulated IL-2(-/-), IL-2Ralpha(-/-), and IL-2Rbeta(-/-) mice are of thymic origin. CONCLUSIONS: Null mutations at IL-2/IL-2Ralpha and beta loci differentially affect intraintestinal and intrathymic T lymphopoiesis. These conditions are associated with lesions of intestinal inflammation that are mediated by thymus-derived T cells.

Animals↗
PubMed2000

The extracellular domain of CD4 regulates the initiation of T cell activation.

To assess the respective contribution of the extracellular and intracellular domains of CD4 in regulating early T cell activation events, we have used a CD4-independent murine T cell clone transfected with human CD4. Stimulation of CD4 positive clones could only be observed if CD4 molecules associated to lck were co-aggregated with the TCR complex, confirming that the simultaneous interaction of MHC class II molecules with the CD4/lck complex and the TCR is required to initiate T cell activation. To assess the involvement of the extracellular portion of CD4 in this process, we transfected a chimeric molecule (EGFRCD4) consisting of the extracellular portion of the epidermal growth factor receptor (EGFR), and of the transmembrane and cytoplasmic domains of human CD4. Although this chimeric molecule associates with lck, transfected clones were induced to proliferate by mAb specific for TCR in the absence of co-aggregation. A new regulatory role for the extracellular domain of CD4 which is independent of its interaction with MHC class II molecules is thus revealed in these experiments. Taken together, our results demonstrate that, in a CD4-independent cell line, two domains of CD4 regulate early T cell activation events: (1) its association with lck and (2) its extracellular domain, independently of its interaction with MHC class II molecules.

Animals↗
PubMed1999

TCR alpha beta-independent CD28 signaling and costimulation require non-CD4-associated Lck.

Whether the sequelae of signals generated through CD28 either directly or in circumstances of costimulation require proximal events mediated by p56lck remains contentious. We demonstrate that CD4-, but not CD4+ clonal variants respond to CD28-specific mAb with both early and late indicators of activation. Forced expression of A418/A420-mutated CD4 or wild-type CD4 in the CD4- variant recapitulated the CD28-mediated responses of the CD4- and CD4+ variants, respectively. The implicated involvement of non-CD4-associated Lck is formally demonstrated by overexpressing S20/S23 Lck or wild-type Lck in CD4+ variants. The former, but not latter, rescues direct CD28 signaling, and supports costimulation. The results demonstrate that constitutive levels of non-CD4-associated Lck functionally limit CD28-mediated signaling.

Animals↗
PubMed1999

CD4-mediated inhibiton of IL-2 production in activated T cells.

The role of CD4 in T cell activation has been attributed to its capacity to increase the avidity of interaction with APC and to shuttle associated Lck to the TCR/CD3 activation complex. The results presented in this study demonstrate that ligation of CD4 inhibits ongoing responses of preactivated T cells. Specifically, delayed addition of CD4-specific mAb is shown to inhibit Ag- or mAb-induced responses of both primary T cells and T cell clonal variants. The Ag responses of the latter are independent of the adhesion provided by CD4; thus the observed inhibition is not due to blocking CD4-MHC interactions. Further, analysis of the clonal variants demonstrates that CD4-associated Lck is not essential for the inhibition observed, as anti-CD4 inhibits responses of clonal variants, expressing a form of CD4 unable to associate with Lck (double cysteine-mutated CD4). The inhibition is counteracted by the addition of exogenous IL-2, demonstrating that the block is not due to a lesion in IL-2 utilization, rather its production. It is demonstrated that the delayed addition of anti-CD4 results in a rapid reduction in steady-state levels of IL-2 mRNA in both primary T cells and clonal variants.

Animals↗
PubMed1999

Speculation on the lineage relationships among CD4(-)8(+) gut-derived T cells and their role(s).

The thymus-independent T lymphopoietic capacity of the murine intestinal mucosa has been established. Cryptopatches have now been identified as the location of the elusive precursors for gut-derived T cells. These cryptopatch cells have been shown to give rise to intestinal T cells expressing either TCRgammadelta or TCRalphabeta. Here we discuss the role of MHC in the development and selection of gut-derived T cells. Through the analysis of iIEL selection in animals expressing a transgenic TCRalphabeta, in the presence or absence of p56(lck), we discuss lineage relationships among CD4(-)8(+) iIEL subsets, and their possible function(s).

Animals↗
PubMed1999

Immobilization of glycosylphosphatidylinositol-anchored proteins inhibits T cell growth but not function.

Accumulating evidence suggests that proteins tethered to the plasma membrane through glycosylphosphatidylinositol (GPI) anchors share common biological properties. In the present study we demonstrate that GPI-anchored proteins regulate T cell growth. Specifically, anti-TCR-induced proliferation was profoundly inhibited by co-immobilized mAb specific for Thy-1, CD48 and Ly6A/E. However, neither IL-2 production nor the effector function of cytotoxic T lymphocytes was impaired in these circumstances. Analysis of the IL-2 receptor (IL-2R) signaling pathway revealed that the association of IL-2R beta and gamma chains with the Janus kinases, JAK1 and JAK3, was not perturbed in the presence of mAb specific for GPI-linked proteins. However, in these conditions, IL-2-mediated recruitment of IL-2Ralpha, beta and gamma chains, resulting in the formation of the high-affinity hetero-trimeric IL-2R, was inhibited. The resulting phosphorylation of JAK1 and JAK3, indicative of their activation states, was correspondingly reduced. These results characterize a novel state of T cell physiology in which effector function is maintained, in the absence of clonal expansion. A physiological role for GPI-anchored proteins in the maintenance of cellular homeostasis and function is discussed.

Animals↗
PubMed1998

Interleukin 2-mediated uncoupling of T cell receptor alpha/beta from CD3 signaling.

T cell activation and clonal expansion is the result of the coordinated functions of the receptors for antigen and interleukin (IL)-2. The protein tyrosine kinase p56(lck) is critical for the generation of signals emanating from the T cell antigen receptor (TCR) and has also been demonstrated to play a role in IL-2 receptor signaling. We demonstrate that an IL-2-dependent, antigen-specific CD4(+) T cell clone is not responsive to anti-TCR induced growth when propagated in IL-2, but remains responsive to both antigen and CD3epsilon-specific monoclonal antibody. Survival of this IL-2-dependent clone in the absence of IL-2 was supported by overexpression of exogenous Bcl-xL. Culture of this clonal variant in the absence of IL-2 rendered it susceptible to anti-TCR-induced signaling, and correlated with the presence of kinase-active Lck associated with the plasma membrane. The same phenotype is observed in primary, resting CD4(+) T cells. Furthermore, the presence of kinase active Lck associated with the plasma membrane correlates with the presence of ZAP 70-pp21zeta complexes in both primary T cells and T cell clones in circumstances of responsive anti-TCR signaling. The results presented demonstrate that IL-2 signal transduction results in the functional uncoupling of the TCR complex through altering the subcellular distribution of kinase-active Lck.

Animals↗
PubMed1998

lck-independent inhibition of T cell antigen response by the HIV gp120.

Binding of the HIV envelope glycoprotein gp120 to CD4 inhibits T cell activation. We have used a murine T cell clone transfected with either wild-type human CD4 or mutated forms of CD4 to characterize the pathways involved in this inhibitory effect of gp120. Ag-induced proliferation of T cell clones transfected with human CD4 was completely inhibited in the presence of gp120, even though stimulation of this clone is independent of a CD4/MHC class II interaction. In addition, our results demonstrate that the inhibition by gp120 is not due to the sequestration of lck from TCR and does not require activation of lck by gp120. This suggests that CD4 can regulate the initiation of T cell activation independently of its interaction with lck. Moreover, we demonstrate that the nonresponsiveness induced by gp120 can be reversed by soluble CD4 when added early after onset of stimulation and that gp120 exerts its inhibitory effect when cells are in the G0 > or = 1 phase of the cell cycle.

Animals↗
PubMed1997

Physical association of CD4 and CD45 in primary, resting CD4+ T cells.

CD45 is a family of transmembrane protein tyrosine phosphatases that are essential to T lymphocytes' responses to antigen-receptor stimulation. It is involved in the regulation of Src-family protein tyrosine kinases, Lck and Fyn. The object of this study was to determine how CD45 molecules are directed to such substrates at the antigen-receptor complex upon stimulation of resting T cells. We demonstrate that CD45 is physically associated with CD4 in resting, primary lymph node T cells. Further, CD4-dependent, antigen-mediated activation of primary CD4+ T cells results in disruption of CD4-CD45 complexes, suggesting a role for these complexes in the activation process. Moreover, CD45 coprecipitates with CD4 molecules which are associated with Lck, as well as with those which are not associated with Lck. Consistent with these observations and the role of CD45 in the regulation of Lck function, effects on CD4-associated membrane Lck are demonstrable. Since antigen presentation by MHC class II results in the coaggregation of CD4 with the antigen-receptor complex, the association described in this study provides a physical basis through which CD45 could be included.

Animals↗
PubMed1995

T-cell development and selection in the intestinal epithelium.

Though it is now well established that T lymphocytes develop in the murine intestinal epithelium, many features of T lymphopoiesis occurring at this site remain controversial and poorly understood. One of the most contentious issues is whether all TcR alpha beta+ iIEL subsets, characterized by the differential expression of CD4, CD8 alpha and CD8 alpha, develop in situ, as suggested by the analysis of athymic radiation chimeras and parabionts. Athymic radiation chimeras have also been used to study positive and negative selection events imposed on developing iIEL expressing an MHC class I restricted transgenic TcR alpha beta. Results reveal that while distinct mechanisms may regulate these processes in the gut, the functional repertoire generated at this site is identical to that resulting from intra-thymic T-cell development. Strikingly, this is not the case for the development and selection of iIEL expressing an MHC class II restricted TcR alpha beta. Features peculiar to MHC class II molecules expressed by enterocytes are discussed in this context. In addition, two model systems are presented towards understanding the basis for the observed oligoclonal repertoire of TcR alpha beta+ iIEL. Specifically, the role of luminal antigen is addressed by comparing the repertoire of iIEL developing in the orthotopic gut with that of iIEL developing in an ectopic sterile intestine in the same athymic animal. The role of TcR alpha beta-mediated signals in support of the putative oligoclonal expansion of iIEL, is addressed using mice lacking the protein tyrosine kinase, Lck.

Animals↗
PubMed1994

Distinct roles for CD4 and CD8 as co-receptors in T cell receptor signalling.

We demonstrate that CD4 and CD8 modify signals induced through the T cell receptor for antigen (TCR alpha beta) in distinct fashions. Pretreatment of CD4+ lymph node T cells with CD4-specific monoclonal antibody results in a tenfold inhibition of DNA synthesis induced by anti-TCR alpha beta. In contrast, pretreatment of CD8+ T cells with CD8-specific mAb has no effect on DNA synthesis subsequently induced through TCR alpha beta. While inhibiting late activation signals, pretreatment with anti-CD4 does not detectably alter the pattern of anti-TCR alpha beta-induced tyrosine phosphorylation of cellular proteins, nor subsequent Ca2+ mobilization. The distinct biological consequences of anti-CD4 and anti-CD8 pretreatment correlate with the differential association of their respective ligands with the cellular protein tyrosine kinase, p56lck. While both T cell lineages contain similar levels of cellular p56lck, tenfold more is associated with CD4 than with CD8. This difference is associated with the differential effects of pretreatment with anti-CD4 and anti-CD8 on the distribution and activity of p56lck. Further, antibody-mediated aggregation of TCR alpha beta on CD4+ T cells induces the appearance of a p56lck species with decreased mobility in sodium dodecylsulfate-polyacrylamide gel electrophoresis. This effect is observed in CD4+ T cells exclusively and involves the fraction of p56lck which is not associated with CD4. The results presented here demonstrate that the signalling elements which couple the antigen receptor to second messenger-generating systems are under distinct physical and/or functional constraints in the two T cell lineages.

Animals↗
PubMed1994

Distinct involvement of CD45 in antigen receptor signalling in CD4+ and CD8+ primary T cells.

We demonstrate that pretreatment of primary CD4+, but not CD8+ T cells with anti-CD45 inhibits activation signals induced through the T cell receptor for antigen (TCR alpha beta). Specifically, anti-TCR alpha beta-mediated tyrosine phosphorylation of phospholipase C-gamma 1 is inhibited, and this in turn correlates with the inhibition of subsequent Ca2+ mobilization and DNA synthesis. In marked contrast, none of these activation parameters are affected by anti-CD45 in CD8+ T cells. Perturbation of TCR alpha beta signalling in CD4+ cells is observed in conditions which do not detectably affect the level of CD45 expression, or its membrane distribution. Further, changes in the intrinsic phosphatase activity of CD45 are not detectable. While anti-CD45 ablates TCR alpha beta signalling, anti-CD3 epsilon-mediated activation is unaffected. This suggests that elements of the antigen receptor complex can be functionally uncoupled, and indicates that the requirements for CD45 in signalling through these two elements are different. The results demonstrate that the involvement of CD45 in coupling TCR alpha beta to second messenger-generating pathways is under distinct physical and/or functional constraints in primary CD4+ and CD8+ T cells.

Animals↗
PubMed1994

Class II major histocompatibility complex-restricted T cell function in CD4-deficient mice.

Previously, we and others have demonstrated that CD4-deficient mice have a normal number of T cells and B cells with a significant population of CD4-8-TcR alpha beta+ T cells. Surprisingly, however, these mice lacking CD4 show in vivo immunoglobulin isotype class switching from IgM to IgG in response to sheep erythrocytes and vesicular stomatitis virus. In this study we have depleted various subpopulations of T cells in vivo and shown that the population of CD4-8-TcR alpha beta+ T cells is responsible for providing "help" in the antibody response of CD4-deficient mice to vesicular stomatitis virus infection. We have used antigen-specific proliferation assays and blocking studies with class I and II major histocompatibility complex (MHC)-specific purified antibodies to show that these cells are class II MHC-restricted in responses against the T cell-dependent antigen keyhole limpet hemocyanin (KLH). Finally, phenotypic analysis of the CD4-CD8- thymocytes in CD4-deficient mice shows that these cells have a more mature phenotype than the CD4-8- thymocytes in wild type mice. These results indicate that CD4 is not absolutely necessary for positive selection or effector function of class II MHC-restricted helper T cells.

Animals↗