Biomedical subjects

Z Brahmi

Publications and source records attributed to Z Brahmi.

At least 37 records · Page 2Linked to original sources

PubMed1995

Upregulation of B7 molecules by the Epstein-Barr virus enhances susceptibility to lysis by a human NK-like cell line.

The original human NK-like line YT was reported to lyse K562 and several B- and T-cell lines. The YT subline we are investigating, YT-INDY, does not lyse K562 or the T-cell line Molt-4. It does, however, lyse the EBV+ Burkitt lymphoma (BL) B-cell line Raji and EBV-immortalized B-cell lines. Several EBV- BL lines and an EBV- pre-B-cell leukemia line that we tested were not appreciably lysed by YT-INDY. To determine if EBV plays a role in TC susceptibility to lysis by YT-INDY, we compared YT-INDY's ability to lyse the EBV- BL line BL41 to its ability to lyse an EBV-infected derivative of BL41. The EBV-infected cell line was lysed, on average, at twice the level of the uninfected line. CD28/B7 interactions appeared to be involved in TC recognition by YT-INDY. Therefore, we examined the level of expression of B7 molecules on the infected and uninfected BL41 lines. An average of 15% of the uninfected BL41 cells expressed B7-1/B7-3, compared to 79% of the infected. B7-2 expression was similar in the two cell lines. Lysis of EBV-infected BL41 was reduced by anti-B7-1/B7-3 (BB1) or anti-CD28 antibodies (Abs) to the level of lysis of the uninfected line, indicating that upregulation of B7-1/B7-3 by the virus may be responsible for the enhanced susceptibility. We attempted to determine the particular EBV latent protein responsible for B7-1/B7-3 upregulation by analyzing BL41 clones expressing LMP1, EBNA-2/EBNA-LP, or EBNA-1. All of the high-expressing clones showed a higher level of B7-1/B7-3 expression than the vector-transfected control cell line, with LMP1-expressing clones expressing the highest amount. EBNA-1 clones and a high-expressing EBNA-2/EBNA-LP clone had a slightly higher density of B7-2 on their surface than the remaining clones. The increased expression of molecules of the B7 family correlated with increased susceptibility of the clones to lysis by YT-INDY. Anti-CD28 or a combination of anti-B7-1/B7-3 and anti-By-2 did not inhibit lysis of the clones to the level of lysis of the vector-transfected control cell line in all cases. We conclude that intact EBV enhances susceptibility to YT-INDY lysis by upregulating B7-1/B7-3. EBV proteins expressed individually also enhance susceptibility to lysis and upregulate members of the B7 family.(ABSTRACT TRUNCATED AT 400 WORDS)

B-Lymphocytes↗

PubMed1995

Synergistic inhibition of human cell-mediated cytotoxicity by complement component antisera indicates that target cell lysis may result from an enzymatic cascade involving granzymes and perforin.

A widely accepted theory of lymphocyte-mediated cytotoxicity (CMC) proposes that upon effector cell (EC) and target cell (TC) interaction, release of perforin, serine proteases and other lytic moieties contained within cytoplasmic granules results in TC lysis. Complement activation and the activation of the various enzymatic activities associated with cytotoxic granules have strikingly similar modes of action and both lead to pore formation in their respective targets. We report here that by using antisera to early and late complement components we were able to inhibit CTL, NK and ADCC cytotoxicity up to 100%, even though binding of EC to TC was unaffected. Furthermore, we showed that addition of C1q or C1s (two serine proteases) antisera to C9 antisera, at titers too low to inhibit separately, resulted in synergistic inhibition of CMC. Anti-C1s together with anti-C1q (or anti-C8 with anti-C9) did not result in synergy. This finding supports a cascade model of activation for lytic molecules released from EC. In addition, we demonstrated that anti-C1q and anti-C1s bind to proteins in the 30-kD region and anti-C9 binds to proteins in the 70-kD region, coinciding with published molecular weights of granzymes and perforin, respectively. Finally, lytic ability of purified granules was also inhibited by complement antisera, further suggesting that activation occurs outside of TC. Taken as a whole, these data indicate that TC lysis may be the result of a cascade of events involving granzymes and perforin, analogous to that seen with the complement system.

Antibodies, Monoclonal↗

PubMed1994

Target cell-induced inactivation of cytolytic lymphocytes. Role and regulation of CD45 and calyculin A-inhibited phosphatase in response to interleukin-2.

Cytolytic T lymphocyte (CTL) and natural killer cells (NK) lose their lytic potential after interaction with sensitive target cells that can be restored upon incubation with interleukin-2. In this study we observed that preincubation with Ser/Thr phosphatase inhibitor calyculin A inhibited both CTL and NK cell-mediated cytotoxicity (CMC) in a dose-dependent manner. In contrast, okadaic acid inhibited only CTL-CMC without significantly affecting NK-CMC. Incubation of CTL and NK cells with their sensitive TC inhibited both CTL-CMC by 74% and NK-CMC by > 80%. This loss in lytic activity was accompanied by a loss of 60% and > 80% in the cellular p-nitrophenyl phosphate phosphatase (pNPPase) activity in CTL and NK cells, respectively. When treated with 100 units/ml interleukin-2 for 16-18 h at 37 degrees C, inactivated CTL and NK cells recovered 70% and 100% of their lytic activity and approximately 60% and 100% of phosphatase activity, respectively. Analysis revealed that > 80% of the pNPPase activity was associated with membrane-bound CD45, and it is this phosphatase activity that was reversibly affected by target cell-induced inactivation/reactivation of CTL and NK cells. These results suggest that Ser/Thr phosphatases and CD45 play a key role in modulating the lytic activity of effector cells exposed to sensitive target cells.

Adult↗

PubMed1994

Human granzyme B is essential for DNA fragmentation of susceptible target cells.

We have partially characterized the granules of the human NK cell line, YT-INDY, and assessed granule-mediated lysis and DNA fragmentation of assorted targets. Biochemical studies demonstrated significant quantities of granzyme B (asp-ase) and a heretofore undescribed chymase but no tryptase (i.e., granzyme A or 3) or distinct met-ase. YT-INDY expressed mRNA for granzyme B, perforin and CCPX. The existence of perforin was confirmed by immunoblot. The granules lysed both human and murine NK-sensitive and NK-resistant targets. YT-INDY and NK3.3, two human cytotoxic cells, were also lysed. EGTA reduced lysis by only 50%, suggesting that a perforin-independent lytic pathway is associated with the granules. In addition, 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF), an inhibitor that selectively blocked the chymase and 3,4-dichloroisocoumarin (DCI), an inhibitor that inactivated both chymase and asp-ase activities, marginally affected lysis. By gel electrophoresis and 125I-labeled deoxyuridine release assay, only murine cells (SP2/0 and YAC-1) underwent DNA fragmentation, and cleavage was completely inhibited by DCI, whereas EGTA, AEBSF and aurintricarboxylic acid (ATA) had no effect. The results, therefore, underscore the central role of granzyme B in granule-mediated DNA fragmentation, emphasize that the protease acts via an ATA-resistant endonuclease pathway and stress that nucleolysis does not invariably accompany granule-mediated cytolysis. Finally, ATA inhibited the asp-ase activity of isolated but not granule-associated granzyme B. ATA, therefore, is not a specific endonuclease inhibitor and results obtained with ATA should be viewed cautiously.

Animals↗

PubMed1994

The lack of NK cytotoxicity associated with fresh HUCB may be due to the presence of soluble HLA in the serum.

Human bone marrow transplantation is becoming more common in the treatment of certain forms of cancer despite the scarcity of HLA matched donors. Because human umbilical cord blood (HUCB) has been used as a source for stem cells in bone marrow transplantation, and because NK cells appear to be important in graft versus leukemia response, we investigated the lytic activity of freshly isolated HUCB NK cells (HUCB-NK) against tumor targets and their ability to differentiate into LAK cells following stimulation with various cytokines. Although cytotoxicity mediated by fresh HUCB-NK was low compared to that of adult peripheral blood lymphocyte-derived NK cells (PBL-NK), the ability of HUCB-NK to bind to K562 target cells (TC) was similar to PBL-NK. In addition, the PBL-NK cytotoxicity of postpartum mothers was also low compared to that of normal adult PBL-NK. When we incubated HUCB for 18 hr in either IL-2 or IL-12, we boosted the level of HUCB-NK cytotoxicity to approximately the level observed in PBL-NK and increased the level of perforin, granzyme A, and granzyme B mRNA expression. In addition, when we incubated HUCB in IL-2, IL-4, IL-7, IL-12, TNF-alpha, IFN-alpha, IFN-gamma, or TGF-beta for 5 days, we observed that HUCB was capable of generating LAK cells only when incubated with either IL-2 or IL-12. In contrast, IL-2, IL-7, IL-12, TNF-alpha, and IFN-gamma all generated LAK cells from adult PBL. When we added to the medium low-dose IL-2 and irradiated K562 as feeder cells (mini-LAK), we were unable to generate LAK activity from HUCB-NK, whereas we could generate it with PBL-NK cells under the same conditions. Addition of serum derived from HUCB in a 4-hr 51Cr release assay with PBL-NK as the effector cells (EC) and K562 as the TC resulted in a 42% decrease in PBL-NK-mediated cytotoxicity. Although we detected no TGF-beta in HUCB serum, we did detect high concentrations of soluble class I MHC (sHLA). To our knowledge, sHLA has not previously been shown to inhibit NK cytotoxicity, although the expression of class I HLA on the surface of TC has been shown to inhibit NK cytotoxicity. To study further the effect of sHLA on cell-mediated cytotoxicity, we added various concentrations of sHLA to EC mediating NK, ADCC, and CTL activities. All were inhibited in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1994

Target cell-directed degradation of perforin mRNA in CTL: lack of correlation with loss of protein and lytic ability.

We have previously shown that CTL and NK cells rapidly down regulate perforin mRNA and become functionally inactive within 4-6 hr after exposure to sensitive target cells (TC). We report here for the first time that CTL also down regulate perforin mRNA upon exposure to resistant, but binding, TC. When three separate human MHC-restricted CTL lines were exposed to resistant TC, perforin mRNA was rapidly degraded. Removal of both extracellular Ca++ and Mg++ prevented perforin message down regulation, whereas removal of Ca++ alone did not, indicating that CTL:TC binding was required. Unlike the response of CTL exposed to sensitive TC, resistant TC did not trigger serine esterase (SE) release, suggesting distinct signalling pathways for perforin mRNA down regulation and granule exocytosis. Moreover, using western analysis, we showed that there was limited (< 10%) perforin protein release after CTL:TC interaction, suggesting that CTL loss of lytic activity after exposure to sensitive TC is not due to massive depletion of perforin. Treatment of CTL with mAb to CD2, CD3, CD2 + CD3, CD8, Class I and LFA-1 did not induce perforin mRNA down regulation. Furthermore, mAb to CD2, CD3, CD8, Class I, Class II, CD54 and LFA-1 did not block TC-mediated perforin mRNA down regulation although lysis of TC was inhibited.

Cell Line↗

PubMed1994

Target cell-directed rapid degradation of TNF-alpha messenger RNA in human cytotoxic T cells.

Previously we have shown that upon exposure to sensitive target cells (TC), human cytotoxic T cells (CTL) and natural killer cells (NK) undergo functional inactivation and specifically degrade mRNA-encoding lytic proteins stored in cytoplasmic granules. These lytic proteins include perforin (PFP) and the serine proteases (granzymes). In this study we show that tumor necrosis factor-alpha (TNF-alpha) mRNA undergoes rapid down-regulation after interaction with TC, in a manner identical to PFP and serine protease mRNA. Both PFP and TNF-alpha messages are down-regulated and maintained at low baseline levels while effector cells (EC) are in contact with TC. Moreover, treatment of CTL with antibodies against CD2, CD3, CD8, and class I could not reproduce TC-directed mRNA down-regulation of TNF-alpha and PFP even though lytic ability was inhibited. Anti-CD2 and anti-CD3, however, markedly induced the expression of TNF-alpha mRNA within 15 min. The increase of TNF-alpha mRNA by anti-CD2 and anti-CD3 was offset by the presence of TC, suggesting that TC supply a negative signal to the CTL resulting in degradation of the TNF-alpha mRNA. Furthermore, treatment of CTL with cycloheximide (CHX) did not prevent PFP message loss and did not allow its recovery, suggesting that de novo protein synthesis is not required for mRNA degradation. In contrast, whereas CHX did not prevent TNF-alpha message loss, CHX allowed recovery of TNF-alpha mRNA within 120 min after TC-directed degradation. Taken as a whole, these data suggest that TC provides a negative regulatory signal that triggers the degradation of TNF-alpha message.

Antibodies↗

PubMed1993

Sulfhydryl reactive phenylarsine oxide inhibits signal transduction in NK and LAK cells: effect on zeta-chain phosphorylation and phosphatidylinositol level.

The specific role of sulfhydryl groups in cell-mediated cytotoxicity (CMC) is still unknown. Here we demonstrate that natural killer cells and lymphokine-activated killer cells, when incubated with phenylarsine oxide (PAO), an organoarsenic compound showed a dose- and time-dependent inhibition of CMC and antibody-dependent cellular cytotoxicity (ADCC). PAO interacted directly with the effector cells (EC) without affecting the target cells (TC) or EC:TC conjugate formation. The loss of cytotoxicity was not due to lack of degranulation or to inhibition of serine esterases in PAO-treated cells. However, PAO inhibited the target-induced down regulation of phosphatidylinositol (PI) level in NK cells indicating that PAO blocked the cytolytic cascade at an early stage, upstream of PI. In addition, PAO also did not affect the disulfide link of the zeta-chain dimers, implicated in signal transduction in cytotoxic lymphocytes but did cause the rapid phosphorylation of the zeta chain. Finally, the effect of PAO on CMC was competitively blocked by dithiothreitol, a dithiol, but not by beta-mercaptoethanol, a mono-thiol. Taken together, these results indicate for the first time how sulfyhydryl groups may regulate CMC and ADCC.

Arsenicals↗

PubMed1993

Regulation of resting and IL-2-activated human cytotoxic lymphocytes by exogenous nucleotides: role of IL-2 and ecto-ATPases.

In this investigation we studied the modulation of human NK- and CTL-mediated cytotoxicity in response to extracellular nucleotides. NK cell-mediated cytotoxicity (CMC) was inhibited in a dose-dependent manner by ATP/ADP, GTP/GDP, and by pentasodium triphosphate (PST), whereas MHC-restricted CTL were inhibited by GTP/GDP and PST, but not by ATP/ADP. Triphosphates were the most potent inhibitors, followed by diphosphates and monophosphates which were the least effective, suggesting that the inhibition was not due to the sugars nor adenosine and guanosine nucleotides, but rather to the increasing negative charges. Cultured CTL, fresh NK cells that had been incubated with IL-2 for 18 hr and IL-2-dependent NK 3.3 cells were all inhibited by GTP, but not by ATP. This differential regulation of fresh NK cells and CTL by exogenous nucleotides is dependent upon the presence of IL-2, but IL-4, IL-6, and IL-8 did not have any effect. Mouse CTL are resistant to ATP presumably because they contain high levels of ecto-ATPases. Different levels of ecto-ATPase activity in human CTL and NK cells may therefore explain the difference in the responses of these effector cells to extracellular nucleotides. To test this possibility we determined the levels of ecto-ATPases in human CTL and NK cells and showed that CTL contained five times more ecto-ATPases than NK cells. Incubation of NK cells with IL-2 or IL-4 did not significantly change the level of ecto-ATPase activity on NK cells. Treatment of NK cells with IL-2 also did not significantly change the substrate specificity of NK-ecto-ATPases toward the extracellular ATP and GTP. Furthermore, treatment of CTL and NK cells with a potent ecto-ATPase inhibitor, 5'-fluorosulfonylbenzoyladenosine (FSBA), did not significantly alter the effect of exogenous nucleotides on the lytic potential of CTL and NK cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗

PubMed1993

Signal transduction in cytotoxic lymphocytes: decreased calcium influx in NK cell inactivated with sensitive target cells.

Our laboratory has previously demonstrated that natural killer (NK) cell-mediated cytotoxicity is protein kinase C (PKC)-dependent and that PKC is translocated from the cytoplasm to the plasma membrane during NK cell activation. Furthermore, exposuring NK cells to a sensitive target cell for 4-6 hr at 37 degrees C rendered NK cells functionally inactive and these inactivated effector cells (i) do not turn over PI in response to K562 stimulation and (ii) lose mRNA for perforin and granzyme A and B less than 30 min after contact with K562. In this study, we first confirmed earlier findings that the interaction of sensitive target cells with human NK cells triggers an influx of extracellular calcium into NK cells. In addition, using flow cytometry we demonstrated that there was a delayed maximum uptake of extracellular calcium into functionally inactive NK cells when these cells were reexposed to fresh K562. Finally, we demonstrated that exposuring NK cells to K562 for 4 hr leads to a loss of NK cytotoxic activity and to the maximal expression of CD69.

Antigens, CD↗

PubMed1991

Receptor modulation and early signal transduction events in cytotoxic T lymphocytes inactivated by sensitive target cells.

In previous studies, we demonstrated that NK cells and lymphokine-activated killer cells were inactivated early in the lytic process by susceptible but not by resistant target cells (TC). We examined the functional status of human MHC-restricted CTL, after interaction with sensitive TC. Two CTL lines were generated in vitro by stimulation with irradiated PAMO, an EBV-transformed cell line. CTL were incubated for up to 4 h with an equal number of PAMO, then separated by a SRBC rosette assay. CTL lost greater than 60% of their lytic activity during the first 30 min of incubation, and greater than 90% by 4 h as assessed by their inability to lyse fresh TC. Inactivated CTL had 35% less serine esterase activity than did control CTL. IL-2 restored the lytic potential and serine esterase activity to normal values within 72 h. Exposure of CTL to PAMO for 4 h induced the modulation of 22 to 44% of TCR/CD3, CD4/CD8, and class I Ag from the cell surface. In contrast, the expression of CD69, and class II Ag increased and there was no change in the expression of CD2, CD28, or LFA-1 Ag. Furthermore, early metabolic events that usually follow CTL-ligand interaction such as phosphatidylinositol metabolism and transient increase in intracellular calcium, did not occur in inactivated CTL upon challenge with PAMO. PMA and the calcium ionophore A23187, restored cytolytic activity, indicating that protein kinase C can be activated and translocated in inactivated CTL. Our data suggest that TC-induced inactivation of CTL may be due to the modulation of key membrane molecules and the lack of certain secondary messengers involved in signal transduction.

Antigenic Modulation↗

PubMed1991

Inhibition of the calpain-mediated proteolysis of protein kinase C enhances lytic activity in human NK cells.

Recent evidence from our laboratory has demonstrated that NK/LAK cell activation of human lymphocytes is protein kinase C (PKC)-dependent. Here, we have investigated the translocation of PKC in human NK cells exposed to sensitive targets or to PMA, a phorbol ester. In NK cells exposed to K562 for 6 hr, we observed a weak translocation of PKC whereas in NK cells exposed to PMA more than 90% of cytosolic PKC was translocated to the membrane in less than 5 min. Stimulation of NK cells with an NK-resistant target, however, did not translocate PKC even after 6 hr. Translocation of PKC to the membrane was followed by the appearance of PKM, the cytosolic calcium/phospholipid (Ca2+/PL)-independent form of PKC. The conversion of PKC to PKM was mediated by calpain, an intracellular calcium-dependent thiol proteinase. When we used two inhibitors of calpain, calpain inhibitor I (CI-I) and calpain inhibitor II (CI-II), both caused a dose-related enhancement of NK-CMC when the inhibitors were present throughout the 3-hr chromium release assay. This enhancement could be circumvented by PMA or by the PKC inhibitor H-7. CI-I and CI-II added together caused a greater increase in NK-CMC than when each was added alone. CI-I and CI-II also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), substantiating further our previous contention that the activation of both NK-CMC and ADCC may involve a common lytic pathway. Activation of NK cells with IL-2 for 18 hr at 37 degrees C was inhibited in the presence of CI-I. To investigate a possible feedback inhibition mechanism due to the buildup of PKC, we examined phosphatidylinositol (PI) metabolism in NK cells activated by IL-2 in either the presence or the absence of CI-I. We observed a significant decrease in PI turnover when NK cells, activated in the presence of IL-2 and CI-I, were stimulated with K562 as compared to NK cells activated by IL-2 alone, then stimulated with K562.

Adult↗

PubMed1991

Rapid loss of perforin and serine protease RNA in cytotoxic lymphocytes exposed to sensitive targets.

We have previously reported that cytotoxic lymphocytes, when exposed to sensitive target cells, temporarily lose their lytic potential. The mechanism leading to this loss of lytic activity is still unknown but it is reversible and the lytic potency can be recovered when the effector cells are incubated with interleukin-2 (IL-2) for 12-14 hr. In this study, we have investigated the regulation of RNA coding for perforin and for two serine proteases, HSP1 and HSP2, in cytotoxic lymphocytes exposed to sensitive targets. Perforin and the two serine proteases are contained in granules of major histocompatibility complex (MHC)-restricted and non-MHC-restricted cytotoxic lymphocytes, but their exact role in the lytic mechanism is still debated. Here we used four different human cytotoxic lymphocytes (CTL) as effector cells: an MHC-restricted CTL (SG-CTL), a non-MHC-restricted CTL (IE6), a natural killer (NK)-like cell line (3.3) and lymphokine-activated killer (LAK) cells. In all effector cells we observed a rapid loss of perforin and of serine protease RNAs within 5 min following the addition of sensitive targets. The effector cells recovered the RNA messages as early as 30 min, although the kinetics of recovery was faster with CTL than with NK-like or LAK effector cells. When we exposed the effector cells to resistant targets we did not detect any loss of perforin or serine protease RNAs. Incubation of the effector cells with cycloheximide, prior to the addition of sensitive targets, did not block message loss, indicating that de novo protein synthesis was not required in this process. Cycloheximide treatment, however, inhibited the recovery of perforin and serine protease RNAs. Taken together, our results indicate that the target-mediated loss of lytic activity in cytotoxic lymphocytes may be a consequence of the down-regulation of perforin or of serine protease transcripts, or both.

Blotting, Northern↗

PubMed1991

Mouse targets undergo double-strand DNA fragmentation when exposed to syngeneic or xenogeneic LAK cells whereas human targets undergo single-strand breaks.

A number of recent studies have shown that mouse target cells (TC) of hematopoietic origin, when exposed to cytotoxic lymphocytes, undergo double-stranded DNA fragmentation. The cause and relevance of the fragmentation remain controversial. In this study we generated a number of mouse (M-LAK) and human LAK (H-LAK) cells and exposed them to a variety of mouse and human TC. YAC and SP/2, 2 mouse TC underwent rapid and extensive fragmentation when lysed by either human or mouse LAK whereas K562 and Daudi, 2 human TC, under the same conditions did not. All 4 TC, however, were killed quite efficiently. Next we labeled TC with 125I-deoxyuridine, exposed them to LAK cells for up to 18 h and loaded the LAK:TC mixtures over an alkaline linear sucrose gradient. After lysing the cells with a lysis buffer containing Triton X-100 we showed that K562 that had been in contact with LAK cells for more than 1 h exhibited single-strand nicks. However, whereas double-strand fragmentation preceded chromium release (lytic activity), the appearance of single-strand nicks did not. Finally, protein synthesis was not required for either type of fragmentation. In summary, we have demonstrated that: (1) the ability to undergo DNA fragmentation is a property of the TC rather than the effector cells that mediated their death, and (2) K562 and Daudi, 2 human TC, undergo single-strand nicks when lysed by LAK cells whereas SP/2 and YAC, 2 mouse TC undergo double-strand fragmentation when exposed to the same syngeneic or xenogeneic effector cells.

Animals↗

PubMed1989

Target cell-directed inactivation and IL-2-dependent reactivation of LAK cells.

In a previous study, we demonstrated that human natural killer cells (NK) lost their lytic activity after interaction with a sensitive target. The loss of NK activity also led to the loss of antibody-dependent cellular cytotoxicity (ADCC), prompting us to postulate that NK and ADCC activities may result from a common lytic mechanism. In this study, we examined whether nonadherent lymphocytes cultured 7 days in the presence of IL-2 (lymphokine-activated killer (LAK) cells) could also be inactivated and, subsequently, be reactivated in the presence of IL-2. We tested three populations of effector cells (EC): cells isolated from freshly drawn blood and tested immediately, cells cultured with IL-2 for 18 hr, and LAK cells. Once they have interacted with K562, all three cell populations lost greater than 90% of their NK-like lytic activity (NK-CMC) but only 80% of ADCC. However, when we treated the three cell types with antibody-coated K562, they lost 90-99% of NK-CMC and 90-97% of ADCC. In these inactivated effector cells we also observed: (i) a reduction in membrane expression of C-reactive protein; and (ii) a decrease in the expression of Leu-11a when EC were inactivated with antibody-coated K562. The loss of lytic activity against K562 was accompanied by a concomitant loss of activity against other LAK-sensitive targets as well as against antibody-coated targets (ADCC). In competitive inhibition experiments the inactivated effector cells failed to inhibit normal NK-CMC and ADCC activities mediated by fresh NK cells. As we have shown previously, this target-directed inactivation was not due to cell death or to lack of conjugate formation. Inactivated LAK cells regained their lytic potential when cultured with IL-2 and this effect was time dependent. By 72 hr, LAK cells inactivated with K562 regained 99% NK-CMC and 82% ADCC, whereas LAK cells inactivated with antibody-coated K562 regained only 80% NK-CMC and 70% ADCC. When we treated the effector cells with emetine, a potent inhibitor of protein synthesis, we could still inactivate the effector cells with K562 and with antibody-coated K562 but could not reactivate them with IL-2.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1989

Low dose total body irradiation: a potent anti-retroviral agent in vivo.

Immunosuppression characterizes many human diseases including leukemia and AIDS. Friend virus (FV)-induced murine leukemia is a useful model for studying both malignancy and immunosuppression. In a previous series of experiments, we have demonstrated that untreated FV-infected mice died within 40 days post-infection, whereas infected mice given 150 cGy total body irradiation (TBI) on days 5 and 12 exhibited long-term survival. In this report, we show that no leukemic cells or type C virus particles are found in the spleens of mice treated with TBI. In addition, both NK activity as well as bone marrow cell's proliferative responses to PHA and Con A were fully restored. This treatment produces long term control of FV-induced murine leukemia, and thus might have relevance for the treatment of a number of immunosuppressive diseases including AIDS.

Animals↗

PubMed1989

Human IgG1 and mouse IgG3 but not monomeric IgM or IgE facilitate antibody-dependent cell-mediated cytotoxicity by human natural killer cells.

Antibody-dependent cell-mediated cytotoxicity depends upon the interaction of target-cell-bound immunoglobulins with the Fc receptor expressed on different effector cells, notably natural killer (NK) cells. Here, we first generated over 1,500 mouse monoclonal antibodies against Raji, an NK-resistant human target cell. We confirmed IgG3 as the most efficient mouse immunoglobulin for the mediation of human NK ADCC and demonstrated pentameric IgM to be a nonmediator. Since the inability of IgM to mediate ADCC could have been due to steric hindrance secondary to the large size of IgM, we reduced and alkylated pentameric IgM and obtained a high percentage of monomers. These monomers did not mediate ADCC either, although they too promoted binding between NK cells and the Raji targets. Further, IgE anti-trinitrophenyl (TNP) antibodies were unable to mediate human NK ADCC of TNP-coated Raji cells. Next, we selected highly sensitized renal patients whose sera contained high titers of HLA and non-HLA antibodies. We demonstrated that human IgG1 facilitated the mediation of ADCC by human NK cells whereas human IgG3 and IgM did not.

Animals↗

PubMed1988

Inhibition of human natural killer cell activity by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine is an early but post-binding event.

Recent evidence has demonstrated a protein kinase C (PKC)-dependent step in cytotoxic T lymphocyte activation. Here, we examined the influence of PKC in the lytic response of human NK cells to K562, an NK-sensitive tumor target cell. We used the known protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and HA1004. H-7 caused a dose-related inhibition of NK cell-mediated cytolysis (CMC) when the inhibitor was present throughout the course of the 3-h chromium release assay. The 50% inhibitory concentration for H-7 was 7 microM. In contrast, HA1004, which exerts a greater inhibitory effect on cyclic nucleotide-dependent protein kinases than PKC, had no effect on NK-CMC. The suppression of NK-CMC by H-7 was not due to inhibition of binding of the effector cells to target cells and could be reversed by the addition of PMA. H-7 was most effective in abrogating NK-CMC when added to the assay within the first 30 min and treatment of the effector and target cells with H-7 resulted in no loss of NK-CMC. Because nearly 50% of the normal NK lytic activity had taken place by 30 min, this suggested that H-7 inhibited an early event. H-7 exerted a dose-related suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) suggesting that NK-CMC and ADCC share the utilization of PKC, however, HA1004 did not inhibit ADCC. Treating NK cells with IL-2 or IFN-beta did not overcome the inhibition of NK-CMC by H-7. In this study, we have thus demonstrated the presence of a PKC-dependent step in NK-CMC and ADCC.

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