PubMed Health⌕ Search

Biomedical subjects

E T Bloom

Publications and source records attributed to E T Bloom.

At least 37 records · Page 2Linked to original sources
PubMed1998

Redox-sensitive events in Fas-induced apoptosis in human NK cells include ceramide generation and protein tyrosine dephosphorylation.

We previously reported that intracellular oxidation-reduction (redox) regulates NK cell functions and that IL-2-activated NK cells undergo apoptosis upon contact with NK-sensitive target cells. We now report that apoptosis in activated human NK cells is also regulated by redox. Thiol deprivation increased apoptosis in NK cells induced by anti-Fas mAb or Fas ligand-transfected cells, and pretreatment of cells with N-acetyl cysteine, which increased intracellular glutathione, partially inhibited the apoptosis and reversed the effect of thiol-deficient medium, suggesting that Fas-induced apoptosis in NK cells is also redox sensitive. Thiol deprivation did not alter cell surface Fas expression, but did increase ceramide generation following Fas engagement. Although exogenous ceramides induced apoptosis of NK cells, thiol depletion had no effect on this apoptosis. Thiol deprivation increased CPP32 activation induced by Fas engagement, but not by ceramides. These findings suggest that, if ceramide is required for Fas-induced apoptosis, thiol deprivation affects the Fas-mediated signaling pathway at the generation of ceramide and/or upstream thereof. Though tyrosine phosphorylation following Fas engagement was not significantly affected by thiol deprivation, tyrosine dephosphorylation was delayed, suggesting that tyrosine phosphatases may also be redox sensitive. The notion that dephosphorylation is important in the Fas signaling pathway is supported by the finding that tyrosine phosphatase inhibitors significantly enhanced both CPP32 activity and apoptosis following Fas ligation. We conclude that events downstream of tyrosine phosphorylation and upstream of CPP32 activation, including tyrosine dephosphorylation and possibly ceramide generation, are sensitive to regulation by redox in human NK cells, requiring a reducing environment for optimal protection from apoptosis induced by Fas ligation.

Acetylcysteine↗
PubMed1997

Involvement of protein tyrosine kinase p72syk and phosphatidylinositol 3-kinase in CD2-mediated granular exocytosis in the natural killer cell line, NK3.3.

The granular exocytosis pathway is one mechanism by which NK cells and CTLs induce cytolysis of target cells. Triggering through adhesion molecules such as CD2 and LFA-1 as well as Fc gammaRIII (CD16) can invoke this pathway. CD2 is a cell surface glycoprotein present on CTLs and NK cells that plays an important role in both cellular adhesion and signal transduction. Here we report that cross-linking of CD2 as well as CD16 by immobilized Abs enhances granular exocytosis in an NK cell line, NK3.3. Herbimycin, a protein tyrosine kinase (PTK) inhibitor, or wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI 3-K), inhibited completely or almost completely CD2- or CD16-mediated granular exocytosis, suggesting the involvement of protein tyrosine kinases and PI 3-K in both CD2- and CD16-mediated granular exocytosis. We also observed that cross-linking of CD2 as well as CD16 enhances p72syk tyrosine kinase activity, and this enhancement correlated well with the increased tyrosine phosphorylation of several cellular proteins, including the adapter protein Shc. Furthermore, we have observed that cross-linking of CD2 as well as CD16 enhances the PI 3-K activity associated with the tyrosine-phosphorylated cellular proteins and Shc. These results provide insight into the signaling pathways by which triggering of CD2 and CD16 on NK cells leads to cytolysis of target cells.

Androstadienes↗
PubMed1997

Control of cell cycle progression in human natural killer cells through redox regulation of expression and phosphorylation of retinoblastoma gene product protein.

Using thiol deprivation, we have previously shown that the response of natural killer (NK) cells to interleukin-2 (IL-2) is subject to redox regulation downstream of IL-2 binding and internalization. We have now used the IL-2-dependent cell line, NK3.3 to study redox regulation of NK cells further, and found that NK3.3 cells neither incorporated [3H]-thymidine nor completed the G1-S phase transition in medium lacking the thiol-related compounds, L-cystine, and glutathione, despite the presence of sufficient IL-2. Thiol deprivation did not alter the induction of DNA interferon-gamma activated sequence (GAS)-binding activity in response to IL-2. However, the retinoblastoma gene product (RB), a cyclin-dependent kinase (CDK) substrate, was phosphorylated within 24 hours after IL-2 stimulation in standard medium, but its expression and phosphorylation were reduced in thiol-depleted medium in both NK3.3 cells and freshly isolated NK cells. These reductions were not associated with an increased level of p27Kip1, an inhibitor of CDKs CDK6/2 in association with G1 cyclins. Reducing agents, N-acetylcysteine, reduced glutathione or 2-ME restored both RB phosphorylation and DNA synthesis in thiol-deprived NK3.3 cells. The in vitro kinase activities of CDK6 and CDK2 were prematurely increased by thiol deprivation. This enhancement was associated with CDK hyperphosphorylation and prolonged phosphorylation, and could be observed before and beyond IL-2 stimulation. The data suggest the possibility that the premature and prolonged enhancement of CDK activity in thiol-deprived NK cells is associated with, and therefore may contribute to, the reduced expression and phosphorylation of RB, and the associated cell cycle arrest.

Cell Cycle↗
PubMed1996

Differential utilization of Janus kinase-signal transducer activator of transcription signaling pathways in the stimulation of human natural killer cells by IL-2, IL-12, and IFN-alpha.

IL-2-, IL-12-, and IFN-alpha-mediated signaling pathways were analyzed in primary NK cells and in the NK3.3 cell line. Gel mobility shift and immunoprecipitation analyses revealed that in addition to activating STAT3 (signal transducer and activator of transcription-3) and STAT5, IL-2 induced tyrosine and serine phosphorylation of STAT1 alpha, which formed IFN-gamma-activated sequence-binding complexes by itself and with STAT3. Although IL-2 and IFN-alpha activated STAT1 alpha and STAT5, IL-2 predominantly activated STAT5, while IFN-alpha predominantly activated STAT1 alpha. IL-2 induced less STAT1 alpha activation and IFN-alpha induced greater STAT5 activation in NK3.3 cells compared with preactivated primary NK cells. In NK3.3 cells, IL-2 induced comparable formation of c-fos promoter sis-inducible element IFN-gamma-activated sequence-binding complexes containing STAT3 alone with complexes containing STAT3 and STAT1 alpha, while in preactivated primary NK cells, it preferentially induced complexes containing STAT3 and STAT1 alpha. Thus, signaling in NK3.3 cells is not always identical with that in primary NK cells. In contrast to IL-2 and IFN-alpha, IL-12 induced strong tyrosine phosphorylation of STAT4 and variable weak phosphorylation of STAT3. However, supershift analyses using the c-fos promoter sis-inducible element probe showed that IL-12 activated STAT4, STAT1 alpha, and STAT3, and induced complexes containing STAT4 only, STAT4 with STAT1 alpha, STAT3 with STAT1 alpha, or STAT1 alpha only in preactivated primary NK cells. STAT1 alpha activation by IL-12 correlated with increased phosphorylation of serine, but not tyrosine. Finally, IL-2 induced tyrosine phosphorylation of JAK1 and JAK3, while IL-12 induced phosphorylation of JAK2 and TYK2 in both preactivated primary NK and NK3.3 cells. Differential phosphorylation and consequent differential activation of both separate and overlapping STAT proteins by IL-2, IL-12, and IFN-alpha may provide a molecular basis for the similarities and differences in the actions of these cytokines on NK cells.

Base Sequence↗
PubMed1996

Target cell-induced apoptosis of interleukin-2-activated human natural killer cells: roles of cell surface molecules and intracellular events.

We previously reported that natural killer (NK)-sensitive target cells, K562, kill interleukin-2-stimulated (lymphokine-activated killer [LAK]) but not unstimulated NK cells. We have now investigated the molecular basis of this phenomenon. Soluble monoclonal antibody (MoAb) to CD18 inhibited 75% of K562-induced DNA fragmentation and membrane disruption, whereas blocking MoAb to Fas partially inhibited only the DNA fragmentation. MoAbs to CD2, CD11a, CD11b, B7, or CD16 had limited or no effect on K562-induced death of LAK cells. Receptor ligation with either immobilized MoAb to CD18 or Fas induced membrane disruption and DNA degradation in LAK cells independently of K562, and MoAb to CD18, CD11a, or CD11b enhanced DNA fragmentation induced by anti-Fas. Fas-L-transfected Raji cells also killed LAK cells, but only if Fas-L expression was amplified. K562 cells rapidly triggered protein phosphorylation in LAK cells, and the tyrosine kinase inhibitor, Herbimycin A, inhibited DNA fragmentation and membrane disruption. Protease inhibitors strongly suppressed K562-mediated DNA fragmentation of LAK cells, but not membrane disruption. In conclusion, (1) K562-induced death of LAK cells involves primarily CD18, although other molecules, such as Fas, may also be involved; (2) K562-mediated apoptosis of LAK cells requires tyrosine phosphorylation and protease activity; (3) engagement of Fas by immobilized MoAb or Fas-L on target cells can also kill LAK cells; and (4) Fas-immobilized MoAb synergizes with coimmobilized MoAb to CD11a, CD11b, or CD18 for LAK cell killing. Activation-induced death of NK cells may represent a mechanism for NK cell regulation.

Antibodies, Monoclonal↗
PubMed1996

Target-induced death by apoptosis in human lymphokine-activated natural killer cells.

Activated human natural killer (NK) cells undergo rapid apoptotic cell death after ligand binding to the Fc receptor (CD16). We examined whether human NK cells die after engagement in cytolytic functions. Peripheral blood NK cells, with and without prior activation in vitro with interleukin-2 (IL-2), were tested for the occurrence of cell death after incubation with K562, the prototype NK-sensitive target cell. A proportion (15.2%) of NK cells that were stimulated for 3 days with IL-2 and then incubated for 4 hours with K562 cells showed rapid cell death, but NK cells not stimulated with IL-2 did not. This cell death was found to involve nuclear condensation and fragmentation and DNA cleavage, all of which are characteristic of apoptosis. These data indicate that a proportion of activated human NK cells undergo apoptosis as they engage in target cell lysis. Target-induced NK cell death may represent an important mechanism for regulation of inflammatory processes involving NK cells.

Apoptosis↗
PubMed1996

IL-12 administered in vivo to young and aged mice. Discrepancy between the effects on tumor growth in vivo and cytotoxic T lymphocyte generation ex vivo: dependence on IFN-gamma.

Because IL-12 restores allogeneic cytotoxic T lymphocyte (CTL) activity by T cells of aged mice in vitro, we initially assessed whether IL-12 could overcome age-related deficits when given to aged mice in vivo. Growth of P815(H-2(d)) was enhanced in aged compared with young BALB/c (H-2(d)) mice and tumor growth was curtailed by IL-12 in both age groups. Unexpectedly, secondary CTL stimulated ex vivo with P815 were reduced in IL-12-treated mice compared with controls. Primary CTL generated ex vivo across MHC differences in IL-12 treated BALB/c and C57BL/6 young mice were reduced by 90-99%, were dose- and time-dependent, and were associated with reduced allo-stimulated NK-like activity and [3H]thymidine incorporation. IFN-gamma was elevated in sera and in supernatants from allo-stimulated cultures from IL-12-treated mice, while IL-4 was reduced in such supernatants, suggesting that, despite reduced CTL, IL-12 was associated with increased Th1- and reduced Th2-type cytokine production. IL-12 also induced splenomegaly, primarily due to increased numbers of cells lacking markers of mature T, B and NK cells, or macrophages, or polymorphonuclear leukocyte morphology. IFN-gamma mutant mice exhibited reduced splenic enlargement in response to IL-12, suggesting that the splenomegaly was due, in part, to IFN-gamma production. However, reduced CTL generation was not due entirely to dilution of CTL precursor cells because spleen cellularity and size increased 3-fold while CTL activity decreased 10- to 100-fold, and CTL generation normalized to CD8(+) T effector cells was still significantly reduced in IL-12-treated mice. Interestingly, purified CD4(+) and CD8(+) T cells from IL-12-treated normal mice exhibited greater proliferative and cytolytic activities respectively compared with controls. Thus, effector T cells in IL-12-treated mice were not impaired, but exhibited augmented responsiveness, suggesting that IL-12 induced complex interactions among spleen cell populations and that these effects, in part, are mediated by IFN-gamma.

Adjuvants, Immunologic↗
PubMed1995

Differential effects of interleukin-12 treatment on gene expression by allostimulated T cells from young and aged mice.

Alloantigen stimulation was used to examine the effect of interleukin (IL-12) treatment of stimulated cells from young and aged mice on the expression of mRNAs for perforin and granzyme B, two proteins known to be intimately involved in an important lytic pathway used by CTL, and mRNA for interferon (IFN)-gamma, production of which is highly stimulated by IL-12 As reported previously, IL-12 augmented the lytic activity by cells from both young and aged mice, although the relative increase was greater for the latter. The mRNAs encoding perforin and granzyme B were both marginally enhanced at early time points (for cells from young mice) or throughout the stimulation (for cells from aged mice) following allo-stimulation in the presence of IL-12. The levels of augmentation of these mRNAs was consistent with the augmentation of lytic activity. In contrast, mRNA encoding IFN-gamma was markedly enhanced throughout stimulation in cells from animals of both age groups, corresponding to the more substantial increase in interferon protein in response to IL-12.

Aging↗
PubMed1995

IL-12 synergizes with IL-2 to induce lymphokine-activated cytotoxicity and perforin and granzyme gene expression in fresh human NK cells.

NK-mediated cytotoxicity is regulated by a variety of cytokines and is thought to involve perforin and granzymes. The effects of IL-2 and IL-12 on the expression and activation of cytolysis were examined in freshly isolated human NK cells. A dose-dependent increase in cytolysis of the NK-sensitive target cell, K562, and the NK-insensitive but lymphokine-activated killer (LAK) cell-sensitive target, UCLA-SO-M14, was observed after short term culture of purified human NK cells in either IL-2 or IL-12. Moreover, the two cytokines often synergized to produce augmented lytic activity. A suboptimal dose of IL-2 (60 IU/ml) combined with IL-12 (2 U/ml) could induce lytic activity equal to twice the additive effect of each cytokine alone. Northern analyses revealed time-dependent increases in mRNAs encoding for perforin and granzymes A and B following treatment with IL-2 alone or IL-2 plus IL-12. IL-2 and IL-12 also synergized for the induction of granzyme mRNAs, in that treatment with both cytokines increased mRNA levels approximately 50% above the sum of each cytokine alone, as quantitated by phosphorimage analysis, and normalized to GAPDH gene expression. However, the synergy between IL-2 and IL-12 for the induction of mRNA was less dramatic than for lytic activity. Results of experiments in which cytokine-treated cells were pulsed with actinomycin D indicated that the increased granzyme and perforin gene mRNA levels in response to IL-2, IL-12, or the combination were not due to increased transcript stability. The data suggest that low doses of IL-2 and IL-12 synergize to augment NK- and induce LAK-mediated cytotoxicity and that this increase is associated with enhanced transcription of perforin and granzyme genes in a synergistic fashion.

Cells, Cultured↗
PubMed1995

Defective lymphoid development in mice lacking expression of the common cytokine receptor gamma chain.

The common gamma chain (gamma c) of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors is defective in humans with XSCID. Mice lacking gamma c expression had hypoplastic thymuses; the thymocytes responded to gamma c-independent mitogens, but not gamma c-dependent stimuli. Splenic T cells were diminished at 3 weeks of age, but CD4+ T cells markedly increased by 4 weeks. B cells were greatly diminished in contrast with the situation in XSCID. NK cells, gamma delta intestinal intraepithelial lymphocytes, dendritic epidermal T cells, peripheral lymph nodes, and gut-associated lymphoid tissue were absent. These findings underscore the importance of gamma c in lymphoid development. Moreover, differences in humans and mice lacking gamma c expression indicate species-specific differences in the roles of gamma c-dependent cytokines or in the existence of redundant pathways. These mice provide an important model for studying the pathophysiology provide an important model for studying the pathophysiology of and gene therapy for human XSCID.

Animals↗
PubMed1995

The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15.

To understand the molecular bases for cytokine redundancy and pleiotropy, we have compared the Stat proteins activated in peripheral blood lymphocytes (PBLs) by cytokines with shared and distinct actions. Interleukin-2 (IL-2) rapidly activated Stat5 in fresh PBL, and Stat3 and Stat5 in preactivated PBL. IL-7 and IL-15 induced the same complexes as IL-2, a feature explained by the existence of similar tyrosine-phosphorylated motifs in the cytoplasmic domains of IL-2R beta and IL-7R that can serve as docking sites for Stat proteins. IL-13 Induced the same complexes as IL-4, a finding explained by our studies implicating IL-4R as a shared component of the receptors. These studies demonstrate that a single cytokine can activate different combinations of Stat proteins under different physiological conditions, and also indicate two mechanisms by which distinct cytokines can activate the same Stat protein.

Amino Acid Sequence↗
PubMed1995

Human peripheral blood CD4+ and CD8+ T cells express Th1-like cytokine mRNA and proteins following in vitro stimulation with heat-inactivated Brucella abortus.

Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells.

Base Sequence↗
PubMed1994

Cellular and molecular mechanisms of the IL-12-induced increase in allospecific murine cytolytic T cell activity. Implications for the age-related decline in CTL.

The mechanism(s) by which IL-12 augments CTL generation and the potential for IL-12 to alter the age-related decline in CTL activity were assessed using primary CTL stimulated by allogeneic cells. IL-12 at 2 U/ml enhanced the generation of allospecific CTL response in young adult mice by 1.5- to 5-fold, and optimal augmentation was achieved and maintained with as low as 0.1 U/ml IL-12. In contrast to the enhancement of MHC-unrestricted lytic activity, which was most efficient when IL-12 was added near the end of the 5 day culture period, nearly equivalent augmentation of Ag-specific CTL was induced when IL-12 was added from 1 to 5 days before harvest. The augmentation of CTL by IL-12 was not associated with increased incorporation of [3H]TdR. Increased IFN-gamma production was not required for IL-12 to augment CTL activity because when three different Abs against mouse IFN-gamma were each included in the MLC at concentrations abolishing recoverable IFN-gamma protein, they did not inhibit the IL-12 effect but, under some conditions, further increased CTL activity. Ab to IL-2 also did not alter the IL-12 effect. Depletion of ASGM1+ cells from the responding population before stimulation did not significantly alter the response to IL-12. However, depletion of CD8+ T cells from effector cells obtained after stimulation substantially reduced the IL-12-induced augmentation of CTL. Thus, the results suggested that IL-12 augments CTL activity primarily through an effect on T cells, and the augmentation was associated with an approximate doubling of perforin levels, which was not caused by a significantly increased proportion of CD8+ cells. Finally, IL-12 was effective for moderating the age-related decrease in CTL activity, suggesting a potential role for immunomodulation by IL-12.

Aging↗
PubMed1994

Assessment of alloreactive T cell subpopulations of aged mice in vivo. CD4+ but not CD8+ T cell-mediated rejection response declines with advanced age.

The present investigation explored age-related alterations in T cell populations mediating allospecific responses in vivo. Healthy aged and young H-2b and H-2bxH-2k mice were engrafted with major histocompatibility complex (MHC) class II-disparate bm12 skin, rejection of which requires CD8+ T cells, and MHC class I-disparate bm1 skin, rejection of which requires CD8+ T cells. Aged mice of both genders exhibited prolonged survival of bm12 skin grafts relative to their young counterparts but rejected bm1 skin grafts at a rate equivalent to that of young mice. Consistent with prolonged survival of bm12 skin grafts, markedly diminished levels of Iabm12 CTL activity were elicited from T cells of aged mice in vitro. However, no such decline was observed in the level of Kbm1 CTL from T cells of aged mice. The alterations in Iabm12 allospecific responses were not attributable to quantitative changes in CD4+ T cells of aged mice, and addition of soluble T cell helper factors to response cultures of aged mice did not augment Iabm12 cytotoxic T lymphocytes activity. These data demonstrate that aging fundamentally affects CD4+ T cell-mediated allospecific responses particularly in vivo, and that deficient generation of soluble T cell helper factors alone cannot explain this deficit.

Aging↗
PubMed1994

Does the age-related change in CD44-defined T-cell subsets have functional significance for cytotoxic T lymphocyte generation?

CD44 or Pgp-1 is a transmembrane leukocyte adhesion-related glycoprotein which is often expressed in greater density on the membranes of memory T lymphocytes (CD44hi) compared to naive T cells (CD44lo). The proportion of Pgphi or CD44hi cells among T cells is increased with advancing age. We examined the relevance of this alteration for the age-related decrease in the generation of allospecific CTL activity. The findings confirm the age-related increase in the frequency of CD44hi cells in spleens of aged mice of several strains, but also show interstrain variability in the magnitude of the increase (bm1 > C57BL/6 > BALB/c). In contrast, we found that after allo-stimulation, the proportion of cells bearing the memory phenotype is decreased in cells from aged mice, particularly within the CD8+ T cell subset. To determine if these observations reflected an alteration in the frequency or responsiveness of naive T cells, enriched populations of spleen cells depleted of CD44hi cells were prepared from spleen cells of young and aged mice, and stimulated in mixed lymphocyte culture. Enrichment for cells expressing the naive phenotype did not restore the ability of T cells from aged mice to generate allospecific CTL. Together, these findings suggest that (1) the age-related increase in frequency of splenic T cells expressing memory phenotype and concordant decrease in phenotypically naive cells, does not explain the age-related decrease in the ability to generate primary allo-CTL, and (2) naive cells from aged mice exhibit intrinsically compromised ability to generate CTL in response to primary alloantigenic stimulation.

Aging↗
PubMed1994

Increased processing of lymphocyte function-associated antigen-1 in human natural killer cells stimulated with IL-2.

Previously we reported that surface expression of lymphocyte function-associated antigen-1 (LFA-1), the primary leukocyte integrin on human natural killer (NK) and lymphokine-activated killer (LAK) cells, does not differ between NK and LAK cells. In contrast to surface expression, we now report that much higher levels of both precursor and mature forms of LFA-1 molecules were found relative to MHC class I, another membrane glycoprotein, with metabolic labeling of IL-2-stimulated LAK cells compared with native NK cells. An 85-90 kDa glycoprotein, found in much higher quantities in LAK compared with NK cells, appeared to be a precursor of the 95 kDa beta chain of the beta 2 integrin family in human LAK cells because: (i) pulse-chase experiments using LAK cells demonstrated decreased 35S-labeling of the 85-90 kDa molecule with a concomitant increase in the radioactivity of the mature 95 kDa LFA-1 beta chain, (ii) results of protease treatment revealed that the two molecules share virtually identical peptide maps, and (iii) endoglycosaminidase F treatment of LAK cell lysates immunoprecipitated with antibody against LFA-1 beta resulted in the disappearance of both the 85-90 and 96 kDa LFA-1 beta signals, and appearance of a signal at approximately 76 kDa. Digestion of the same immunoprecipitates with neuraminidase resulted in the disappearance of the 95 kDa signal and revealed a single molecular weight signal corresponding to 85-90 kDa. These data suggest that a core protein of approximately 76 kDa becomes N-glycosylated, perhaps terminally with sialic acid residues, to mature into the 95 kDa form. Moreover, the rate of maturation of LFA-1 was more rapid in LAK than NK cells, with half times of 0.8 versus 1.5 h for the alpha chain and 3.7 versus 4.9 h for the beta chain for LAK versus NK cells respectively. IL-2 treatment of NK cells therefore alters the processing of LFA-1 molecules during the transition to LAK cells, providing a larger intracellular reservoir with which to replenish the surface molecule. Together with our previous observation that LFA-1 is phosphorylated and transduces signal more effectively in LAK than NK cells, the findings support the notion that adhesion molecules contribute to the increased function of LAK cells.

Cells, Cultured↗
PubMed1993

Requirement of thiol compounds as reducing agents for IL-2-mediated induction of LAK activity and proliferation of human NK cells.

Thiol-related compounds, such as L-cystine, 2-ME or reduced glutathione (GSH), are important in many lymphoid cell activation pathways. We investigated their role in IL-2-generated lymphokine-activated killer (LAK) activity in human NK (CD16+ and/or CD56+) cells. Depletion of GSH and L-cystine, but not L-leucine or glycine, from medium used during culture of NK cells with IL-2 inhibited LAK and proliferative activities, whereas IL-2-independent lytic function of NK cells remained intact. Addition of L-cysteine, 2-ME or GSH, but not methylated analogs of these compounds (which cannot function as proton donors) to L-cystine/GSH-depleted medium restored proliferative response of NK cells and LAK generation. In the presence of L-buthionine-(S,R)-sulfoximine, an inhibitor of GSH synthesis, IL-2-induced LAK activity and proliferation of NK cells in medium without L-cystine and GSH, could be restored, at least in part, by addition of GSH, but not 2-ME or L-cystine. Furthermore, intracellular GSH levels were depressed in cells cultured in L-cystine/GSH-depleted medium but could be restored by the addition of 2-ME. The results suggest that (1) L-cystine or thiol containing compounds such as L-cysteine, 2-ME, or GSH are necessary for effective IL-2-activation of human NK cells, (2) these compounds must be functional proton-donors, i.e., reducing agents, implying regulation of the IL-2 activation pathway by oxidation-reduction, and (3) GSH synthesis is necessary for the activation. Experiments were performed to begin to dissect the redox-sensitive step(s) in LAK development. Depletion of reducing agents had no effect on internalization of rIL-2. In contrast, intracellular granzyme A activity was significantly depressed in NK cells cultured with rIL-2 in L-cystine/GSH-depleted medium compared with those cultured in medium in which L-cystine levels had been replenished. The findings suggest that step(s) in the transduction of the IL-2 signal in NK cells, between the internalization of IL-2 and the maturation of the lytic mechanism, are subject to regulation by oxidation-reduction.

Animals↗