Biomedical subjects

A D Keegan

Publications and source records attributed to A D Keegan.

At least 19 recordsLinked to original sources

PubMed2003

Reciprocal expression of TRAIL and CD95L in Th1 and Th2 cells: role of apoptosis in T helper subset differentiation.

Upon activation, naïve T helper cells can differentiate into two major distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), as defined by their effector functions and cytokine secretion patterns. Cytokine milieu and costimulatory molecules have been shown to play an essential role in determining T helper differentiation. However, it is still unclear how the effects of signals of costimulatory molecules and cytokines are exerted during T helper differentiation. We show evidence suggesting that while cytokine signals initiate the differentiation program, the selective action of death effectors determines the end point balance of differentiating T helper subsets. We examined the expression of TNF-related apoptosis-inducing ligand (TRAIL) and CD95L in cloned and in vitro differentiated Th1 and Th2 cells. We found that activation-induced expression of TRAIL is exclusively observed in Th2 clones and primary T helper cells differentiated under the Th2 condition, while the expression of CD95L is mainly in Th1 cells. Furthermore, these two subsets exhibit distinct susceptibilities to TRAIL- and CD95L-mediated apoptosis. Th2 cells are more resistant to either TRAIL- or CD95L-induced apoptosis than Th1 cells. More importantly, both Th1 and Th2 cells could induce apoptosis in labeled Th1 but not Th2 cells. Blocking TRAIL and CD95L significantly enhance IFN-gamma production in vitro. Likewise, young MRL/MpJ-lpr/lpr mice also showed more Th1 response to ovalbumin immunization as compared to MRL/MpJ+/+. Therefore, apoptosis mediated by CD95L and TRAIL is critical in determining the fate of differentiating T helper cells.

Animals↗

PubMed2001

Costimulation of resting B lymphocytes alters the IL-4-activated IRS2 signaling pathway in a STAT6 independent manner: implications for cell survival and proliferation.

IL-4 is an important B cell survival and growth factor. IL-4 induced the tyrosine phosphorylation of IRS2 in resting B lymphocytes and in LPS- or CD40L-activated blasts. Phosphorylated IRS2 coprecipitated with the p85 subunit of PI 3' kinase in both resting and activated cells. By contrast, association of phosphorylated IRS2 with GRB2 was not detected in resting B cells after IL-4 treatment although both proteins were expressed. However, IL-4 induced association of IRS2 with GRB2 in B cell blasts. The pattern of IL-4-induced recruitment of p85 and GRB2 to IRS2 observed in B cells derived from STAT6 null mice was identical to that observed for normal mice. While IL-4 alone does not induce activation of MEK, a MEK1 inhibitor suppressed the IL-4-induced proliferative response of LPS-activated B cell blasts. These results demonstrate that costimulation of splenic B cells alters IL-4-induced signal transduction independent of STAT6 leading to proliferation. Furthermore, proliferation induced by IL-4 in LPS-activated blasts is dependent upon the MAP kinase pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Efficient transduction of murine B lymphocytes and B lymphoma lines by modified adenoviral vectors: enhancement via targeting to FcR and heparan-containing proteins.

Murine lymphocytes are relatively refractory to efficient transfection or retroviral gene transduction. Adenovirus has been used as a vector to transduce a wide variety of cell types. Several advantages of adenoviruses are their ability to transduce non-cycling cells and to transduce the majority of cells in a population. Unfortunately, lymphocytes are not susceptible to infection with conventional adenovirus. Therefore, to express genes efficiently in murine B cells, we tested the ability of genetically modified adenovirus to transduce the beta-galactosidase gene. We found that adenovirus containing polylysine in the fiber knob was able to efficiently transduce lipopolysaccharide (LPS)-activated splenic B cells and the B lymphoma line M12.4.1; greater than 80% of the cells expressed beta-galactosidase activity. However, small resting B cells did not express activity unless treated with LPS after infection. This transduction was mediated by interaction with charged molecules since heparan-sulfate, and to a lesser degree chondroitan sulfate, inhibited the transduction. In addition, adenovirus containing a FLAG epitope in the fiber protein was used to target the FcR expressed on B cells using an anti-FLAG antibody. In the presence of anti-FLAG, the modified adenovirus was able to efficiently transduce LPS-activated B cells and several B cell lymphoma lines. Interestingly, in the absence of anti-FLAG, there was low level transduction in the LPS-blasts and in M12.4.1 that was not inhibited by soluble adenovirus fiber protein or agents that block RGD-integrin interactions. These results demonstrate that modified adenovirus efficiently transduce B lymphocytes which will be critical for targeting genes to normal or malignant B cells.

Adenoviridae↗

PubMed2001

NF-kappa B activation plays an important role in the IL-4-induced protection from apoptosis.

IL-4 alone protects cells from apoptosis by insulin receptor substrate (IRS)-dependent and -independent mechanisms. However, in vivo cells are typically exposed to a number of signals at the same time. To determine the contribution of co-stimulatory signals to the regulation of apoptosis by IL-4, we first analyzed whether tumor necrosis factor (TNF)-alpha, which has been shown to inhibit the activation of IRS-1 by insulin, could modify IL-4 signaling and protection from apoptosis. We found that TNF-alpha cooperates with IL-4 in protecting 32D cells from factor withdrawal-induced apoptosis. This effect was independent of the expression of IRS-1, indicating that this cooperation is via an alternative anti-apoptotic pathway. Moreover, TNF-alpha had no effect on the activation of IRS-1 induced by IL-4. IL-4 enhanced TNF-alpha-induced activation of the transcription factor NF-kappaB. Interestingly, pharmacologic inhibition of NF-kappaB activation or protein synthesis resulted in the induction of cell death that could not be inhibited by IL-4, suggesting that IL-4 cooperates with NF-kappaB to signal protection from apoptosis. Supporting this hypothesis, IL-4 also increased NF-kappaB activation induced by anti-CD3 antibodies in primary T cells and protected them from apoptosis induced by receptor engagement. However, IL-4 was not able to inhibit apoptosis induced by anti-CD3 in T lymphocytes isolated from transgenic mice expressing a dominant-negative form of IkappaBalpha that prevents NF-kappaB activation. Thus, in addition to the previously identified IRS-1 pathway, IL-4-induced protection from apoptosis may also be mediated through cooperation with the NF-kappaB family of transcription factors.

Adjuvants, Immunologic↗

PubMed1999

Cutting edge: effects of an allergy-associated mutation in the human IL-4R alpha (Q576R) on human IL-4-induced signal transduction.

A mutation in the human (hu) IL-4R alpha, Q576R, has been linked with allergy in humans. Increased sensitivity of patients cells with this mutation to IL-4 suggest that a Q576R change enhances IL-4 signaling. To directly test this hypothesis, we analyzed the ability of huIL-4R alpha cDNA bearing the Q576R and Y575F mutations to signal tyrosine phosphorylation, DNA-binding activity, proliferation, protection from apoptosis, and CD23 induction in response to huIL-4 in murine cells. Responses generated by the Q576R and Y575F mutants were similar to those of the wild-type receptor, using various concentrations of huIL-4 and times of stimulation. These results indicate that neither the Q576R nor the Y575F mutations have a significant direct effect on IL-4 signal transduction, and that hypersensitive induction of CD23 in cells derived from human allergy patients may be due to different and/or additional alterations in the IL-4 signaling pathway.

Amino Acid Substitution↗

PubMed1999

The IL-4 receptor: signaling mechanisms and biologic functions.

Interleukin-4 is a multifunctional cytokine that plays a critical role in the regulation of immune responses. Its effects depend upon binding to and signaling through a receptor complex consisting of the IL-4R alpha chain and the common gamma chain (gamma c), resulting in a series of phosphorylation events mediated by receptor-associated kinases. In turn, these cause the recruitment of mediators of cell growth, of resistance to apoptosis, and of gene activation and differentiation. Here we describe our current understanding of the organization of the IL-4 receptor, of the signaling pathways that are induced as a result of receptor occupancy, and of the various mechanisms through which receptor function is modulated. We particularly emphasize the modular nature of the receptor and the specialization of different receptor regions for distinct functions, most notably the independent regulation of cell growth and gene activation.

Amino Acid Sequence↗

PubMed1998

Regulation of apoptosis by tyrosine-containing domains of IL-4R alpha: Y497 and Y713, but not the STAT6-docking tyrosines, signal protection from apoptosis.

IL-4 is a cytokine with important antiapoptotic activity. We have analyzed the role that tyrosine-containing domains within the cytoplasmic tail of IL-4R alpha play in IL-4-mediated protection from apoptosis. 32D cells expressing a wt huIL-4R alpha or one truncated at aa 557 were protected by huIL-4 from apoptosis while cells expressing a receptor truncated at aa 657 were not, suggesting that the carboxyl-terminal domain signals protection from apoptosis. However, changing Y713 within this region to phenylalanine had no effect. To analyze the contribution of tyrosine-containing domains independently, we transplanted regions of the huIL-4R alpha to a truncated form of the huIL-2R beta that could not signal protection from apoptosis. Transplantation of the huIL-4R alpha domains containing Y497 or Y713 partially prevented cell death and together signaled protection from apoptosis in response to IL-2 as well as the wt IL-2R beta. Mutation of Y497 and Y713 to phenylalanine inhibited protection. In contrast, transplantation of the domain containing the potential STAT6-docking tyrosines alone had no effect, yet it inhibited the protection mediated by the other domains. Although IL-4R alpha signals Shc and SH2-containing inositol phosphatase (SHIP) phosphorylation, we could not establish an association between their activation and protection from apoptosis. Taken together, this study suggests that the domains of the huIL-4R alpha containing Y497 and Y713 positively regulate protection from apoptosis while the domain containing the STAT6 docking sites suppresses this protection, and that additional signaling molecules other than insulin receptor substrate-1 (IRS1), Shc, or SHIP may be involved in antiapoptotic signaling.

Animals↗

PubMed1998

A role for the insulin-interleukin (IL)-4 receptor motif of the IL-4 receptor alpha-chain in regulating activation of the insulin receptor substrate 2 and signal transducer and activator of transcription 6 pathways. Analysis by mutagenesis.

The interleukin (IL)-4 receptor alpha-chain (IL-4Ralpha) contains a sequence motif (488PLVIAGNPAYRSFSD) termed the insulin IL-4 receptor motif (I4R motif). Mutation of the central Tyr497 to Phe blocks the tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1) and diminishes proliferation in response to IL-4. Recent data suggest that the I4R motif encodes binding sites for several protein tyrosine binding (PTB) domain-containing proteins such as IRS1 and Shc and potentially for the Src homology 2 domain of signal transducer and activator of transcription 6 (STAT6). To analyze the function of the I4R motif in regulating IL-4 signaling, we changed conserved residues upstream and downstream of the central Tyr to Ala in the human IL-4Ralpha. We analyzed the ability of these constructs to signal the tyrosine phosphorylation of IRS2 and STAT6, the induction of DNA binding activity, and CD23 induction in response to human IL-4 (huIL-4) in transfected M12.4.1 cells. Mutagenesis of residues downstream of Tyr497, such as Arg498 or Phe500, to Ala had no effect on any of these responses, suggesting that the I4R motif may not be important for functional Src homology 2 domain interactions. However, mutagenesis of Pro488 to Ala (P488A) greatly diminished the tyrosine phosphorylation of IRS2 and abolished tyrosine phosphorylation of STAT6, induction of DNA binding activity, and CD23 induction in response to huIL-4. By contrast, a P488G mutant signaled these responses to huIL-4. Mutagenesis of hydrophobic amino acids previously shown to contact the PTB domain of IRS1, Leu489 or Ile491, to Ala had only minimal effects on responses to huIL-4. However, changing both Leu498 and Ile491 to Ala greatly diminished the tyrosine phosphorylation of IRS2 and abolished STAT6 activation. Taken together, these results indicate the important role of the I4R motif in regulating IRS docking and suggest that I4R docking to a PTB domain-containing protein regulates activation of the STAT6 pathway.

Alanine↗

PubMed1998

Regulation of cell growth by IL-2: role of STAT5 in protection from apoptosis but not in cell cycle progression.

Cytokines play an essential role in the regulation of lymphocyte survival and growth. We have analyzed the pathways activated by IE-2 that lead to protection from apoptosis and cell proliferation. IL-2 can act as a long-term growth factor in 32D cells expressing the wild-type human (hu)IL-2R beta. By contrast, cells expressing a truncated form of the huIL-2R beta, which is able to induce Bcl-2 and c-myc expression but not STAT5 activation, were not protected from apoptosis by IL-2; consequently, they could not be grown long term in the presence of IL-2. However, IL-2 promoted cell cycle progression in cells bearing the truncated huIL-2R beta with percentages of viable cells in the G0/G1, S, and G2/M phases similar to cells expressing the wild-type huIL-2R beta. Transplantation of a region from the erythropoietin receptor, which contains a docking site for STAT5 (Y343) to the truncated huIL-2R beta, restored the ability of IL-2 to signal both activation of STAT5 and protection from apoptosis. By contrast, transplantation of a region from the huIL-4R alpha containing STAT6 docking sites did not confer protection from apoptosis. These results indicate that the IL-2-induced cell cycle progression can be clearly distinguished from protection from apoptosis and that STAT5 participates in the regulation of apoptosis.

Animals↗

PubMed1998

Regulation of gene expression, growth, and cell survival by IL-4: contribution of multiple signaling pathways.

Interleukin-4 is a cytokine produced by activated T cells, mast cells, and basophils that elicits many important biological responses[1] (see Tab 1). These responses range from the regulation of helper T cell differentiation[2] and the production of IgE[3] to the regulation of the adhesive properties of endothelial cells via VCAM-1[4]. In keeping with these diverse biological effects, high-affinity binding sites for IL-4 (Kd 20 to 300 pM) have been detected on many hematopoietic and non-hematopoietic cell types at levels ranging from 50 to 5000 sites per cell[5]. This review will focus on the discrete signal transduction pathways activated by the IL-4 receptor and the coordination of these individual pathways in the regulation of a final biological outcome.

Amino Acid Sequence↗

PubMed1997

HMG-I(Y) phosphorylation status as a nuclear target regulated through insulin receptor substrate-1 and the I4R motif of the interleukin-4 receptor.

Interleukin (IL)-4 is a cytokine that regulates both the growth and differentiation of hematopoietic cells. Its ligand binding specificity and important signal transduction mechanisms are conferred by the IL-4 receptor alpha chain (IL-4Ralpha). The I4R is a tyrosine-containing motif within IL-4Ralpha that is critical for proliferative responses to IL-4. Although the I4R also contributes to gene regulation, nuclear targets directly regulated by this motif have not been described. It is shown here that the tyrosine at position 497 in the I4R is critical for regulation of the phosphorylation status of a set of nuclear proteins that includes HMG-I(Y), small non-histone chromosomal proteins involved in the control of gene expression in hematopoietic cell lines. Moreover, IL-4 is unable to induce HMG-I(Y) phosphorylation in insulin receptor substrate-1-deficient cells, and the inhibitor wortmannin completely blocks IL-4 regulation of HMG-I(Y) phosphorylation status but not activation of an IL-4 Stat protein. Taken together, these data indicate that HMG-I(Y) is a nuclear target whose phosphorylation status is regulated through the I4R motif via insulin receptor substrate proteins, independent of activation of the Stat pathway.

Androstadienes↗

PubMed1997

The IL-4-induced tyrosine phosphorylation of the insulin receptor substrate is dependent on JAK1 expression in human fibrosarcoma cells.

It has been shown that IL-4 induces the tyrosine phosphorylation of JAK1 and JAK3 in the majority of hemopoietic cell types, and JAK2 and TYK2 in several other types. However, the significance of this tyrosine phosphorylation in regulating IL-4 signaling has not been shown. To determine whether JAKs play a role in activating a signal transduction pathway different from the classical JAK/STAT pathway, we analyzed the ability of huIL-4 to stimulate the tyrosine phosphorylation of one of its major cellular substrates, the insulin receptor substrate (IRS). Using human fibrosarcoma cell lines with mutations in JAK1, JAK2, and TYK2, we found that expression of functional JAK1, but not TYK2 or JAK2, is essential for IL-4-induced tyrosine phosphorylation of IRS. We also provide evidence that the IRS pathway is independent of STAT-6, showing that JAK1 is essential for activating a STAT-independent pathway.

Fibrosarcoma↗

PubMed1996

IL-4 protects cells from apoptosis via the insulin receptor substrate pathway and a second independent signaling pathway.

Although it has been known for some time that IL-4 protects cells from death via apoptosis, very little is known about the mechanism by which IL-4 exerts this effect. In this report, we have studied the molecular mechanisms of the IL-4-induced prevention of apoptosis in the 32D and B cell systems. IL-3 withdrawal has been shown to induce G1-arrest in 32D cells and subsequent death by apoptosis. We found that overexpression of IRS-1 protected 32D cells from death induced by IL-3 deprivation. IL-4 was able to protect 32D cells from apoptosis in the presence or absence of IRS-1. However, the protection from apoptosis of cells cultured in IL-4 was greater in the presence of IRS-1 and it reached levels comparable to those of cells cultured in IL-3. The IRS-1-dependent prevention of apoptosis is linked to the activation of PI 3'-kinase since wortmannin and LY294002, two inhibitors of PI 3'-K, partially inhibited the prevention of apoptosis mediated by IL-4 in 32D-IRS-1 cells after IL-3 withdrawal but not in 32D cells lacking IRS-1 expression. In addition, we found that the IRS/PI 3'-K pathway is, at least in part, responsible for the prevention of apoptosis by IL-4 in normal splenic B cell cultures. In spite of the ability of murine IL-4 to partially protect 32D cells lacking IRS-1 from apoptosis, human IL-4 was not able to prevent cell death in 32D-IRS-1 cells transfected with the human IL-4 receptor mutated in the insulin-lL-4 receptor motif (14R-motif) at amino acid 497 (Y497F). This mutation has previously been shown to abrogate the tyrosine phosphorylation of IRS-1 by human IL-4. These results demonstrate that IL-4 protects 32D cells from apoptosis by two different pathways, one of them mediated by IRS-1. In addition, these results suggest that the 14R-motif of the IL-4R is linked to both pathways.

Animals↗

PubMed1996

IL-4 function can be transferred to the IL-2 receptor by tyrosine containing sequences found in the IL-4 receptor alpha chain.

IL-4 binds to a cell surface receptor complex that consists of the IL-4 binding protein (IL-4R alpha) and the gamma chain of the IL-2 receptor complex (gamma c). The receptors for IL-4 and IL-2 have several features in common; both use the gamma c as a receptor component, and both activate the Janus kinases JAK-1 and JAK-3. In spite of these similarities, IL-4 evokes specific responses, including the tyrosine phosphorylation of 4PS/IRS-2 and the induction of CD23. To determine whether sequences within the cytoplasmic domain of the IL-4R alpha specify these IL-4-specific responses, we transplanted the insulin IL-4 receptor motif (I4R motif) of the huIL-4R alpha to the cytoplasmic domain of a truncated IL-2R beta. In addition, we transplanted a region that contains peptide sequences shown to block Stat6 binding to DNA. We analyzed the ability of cells expressing these IL-2R-IL-4R chimeric constructs to respond to IL-2. We found that IL-4 function could be transplanted to the IL-2 receptor by these regions and that proliferative and differentiative functions can be induced by different receptor sequences.

Amino Acid Sequence↗

PubMed1995

Similarities and differences in signal transduction by interleukin 4 and interleukin 13: analysis of Janus kinase activation.

The cytokines interleukin (IL) 4 and IL-13 induce many of the same biological responses, including class switching to IgE and induction of major histocompatibility complex class II antigens and CD23 on human B cells. It has recently been shown that IL-4 induces the tyrosine phosphorylation of a 170-kDa protein, a substrate called 4PS, and of the Janus kinase (JAK) family members JAK1 and JAK3. Because IL-13 has many functional effects similar to those of IL-4, we compared the ability of IL-4 and IL-13 to activate these signaling molecules in the human multifactor-dependent cell line TF-1. In this report we demonstrate that both IL-4 and IL-13 induced the tyrosine phosphorylation of 4PS and JAK1. Interestingly, although IL-4 induced the tyrosine phosphorylation of JAK3, we did not detect JAK3 phosphorylation in response to IL-13. These data suggest that IL-4 and IL-13 signal in similar ways via the activation of JAK1 and 4PS. However, our data further indicate that there are significant differences because IL-13 does not activate JAK3.

Animals↗

PubMed1995

Interleukin 4 signals through two related pathways.

The interleukin 4 (IL-4) signaling pathway involves activation, by tyrosine phosphorylation, of two distinct substrates, a signal-transducing factor (STF-IL4) and the IL-4-induced phosphotyrosine substrate (4PS). It is not known whether the IL-4-mediated activation of these substrates occurs via related or distinct signaling pathways. We report that 32D cells, an IL-3-dependent myeloid progenitor cell line in which no phosphorylated 4PS is found, activate high levels of STF-IL4 in response to IL-4. Consistent with the known requirement for 4PS or insulin receptor substrate 1 (IRS-1) in IL-4-mediated mitogenesis, activation of STF-IL4 in 32D cells is not sufficient for IL-4-inducible c-myc expression. In addition, we have examined the ability of 32D cells transfected with different truncation mutants of the human IL-4 receptor to activate Jak-3 kinase and STF-IL4 in response to human IL-4. As in the case of 4PS/IRS-1, we have found that activation of both Jak-3 and STF-IL4 requires the presence of the IL-4 receptor region comprising aa 437-557. The finding that the same region of the IL-4 receptor is required for the induction of both 4PS/IRS-1 and STF-IL4 suggests that the IL-4-stimulated activation of these two substrates might involve common factors.

Animals↗

PubMed1995

Identification of Fc epsilon RIneg mast cells in mouse bone marrow cell cultures. Use of a monoclonal anti-p161 antibody.

A monoclonal hamster antibody (K-1) specific for a 161-kD mast cell surface glycoprotein was derived. p161 is expressed on normal and cultured mast cells and on some macrophages, but not on basophils or other hematopoietic cells. A population of Fc epsilon Rneg cells expressing p161 was found in short term cultures of bone marrow cells grown in interleukin (IL)-3. These cells were purified and propagated for extended periods in IL-3. They express c-kit and Fc gamma RII/III, contain alcian blue-positive granules and histamine, and secrete IL-3 in response to ionomycin treatment. Their morphology is consistent with that of mast cells. We propose that they represent Fc epsilon RIneg mast cells that can be detected and purified because of their p161 expression.

Animals↗

PubMed1995

Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis.

By searching a database of expressed sequences, we identified a member of the signal transducers and activators of transcription (Stat) family of proteins. Human and murine full-length cDNA clones were obtained and sequenced. The sequence of the human cDNA was identical to the recently published sequence for interleukin-4 (IL-4)-Stat (J. Hou, U. Schindler, W.J. Henzel, T.C. Ho, M. Brasseur, and S. L. McKnight, Science 265:1701-1706, 1994), while the murine Stat6 amino acid and nucleotide sequences were 83 and 84% identical to the human sequences, respectively. Using Stat6-specific antiserum, we demonstrated that Stat6 is rapidly tyrosine phosphorylated following stimulation of appropriate cell lines with IL-4 or IL-3 but is not detectably phosphorylated following stimulation with IL-2, IL-12, or erythropoietin. In contrast, IL-2, IL-3, and erythropoietin induce the tyrosine phosphorylation of Stat5 while IL-12 uniquely induces the tyrosine phosphorylation of Stat4. Inducible tyrosine phosphorylation of Stat6 requires the membrane-distal region of the IL-4 receptor alpha chain. This region of the receptor is not required for cell growth, demonstrating that Stat6 tyrosine phosphorylation does not contribute to mitogenesis.

Amino Acid Sequence↗