Generation of Inositol Phosphates during Triggering of Cytotoxicity in Human Natural Killer and Lymphokine-Activated Killer Cells

We investigated early molecular mechanisms involved in the triggering of cytolytic responses in natural killer (NK) and lymphokine-activated (LAK) cells. When NK or LAK cells were conjugated to the sensitive target cells K562, an increased formation of both inositol monophosphate (IP1) and inositol trisphosphate (IP3) was detected. Target cells like Raji or Jok-1, which form conjugates with NK cells but are insensitive to NK lysis, did not elicit IP1 formation. Treatment of NK cells with interleukin 2 increased the basal turnover of inositol phosphates and enhanced the phosphatidyl inositol breakdown upon confrontation with sensitive targets. These finding indicate that hydrolysis of phosphatidyl inositols is associated with the signal which triggers the cytolytic response in NK and LAK cells. These events therefore constitute an early marker of the cytolytic activation.     

Cell-to-Cell Mediated Inhibition of Natural Killer Cell Proliferation by Monocytes and its Regulation by Histamine H2-Receptors

Human peripheral blood monocytes, recovered by counter-current centrifugal elutriation, suppressed interleukin-2 (IL-2)-induced proliferation of autologous lymphocytes, recovered from low density Percoll fractions. Cell sorting experiments, analysis of phenotype of proliferating cells, and removal of defined cellular subsets by complement cytotoxicity revealed that IL-2-induced proliferation was confined to CD3-/16+/56+ natural killer (NK) cells. Monocyte-induced suppression of NK-cell proliferation was completed within 1 h of incubation with monocytes and unrelated to the formation of prostaglandins or other intermediary factors. The biogenic amine histamine, acting via H2-type histamine receptors (H2R) on monocytes, completely counteracted the monocyte-mediated suppression of IL-2-induced NK-cell proliferation. Our data are suggestive of a H2R-regulated, cell-cell-mediated mechanism by which monocytes down-modulate NK-cell proliferation.     

Regulation of Natural Killer Cell Activity by Transforming Growth Factor-β and Prostaglandin E2

The separate and combined effects of transforming growth factor-β1 (TGF-β1) and prostaglandin E₂ on human natural killer (NK) activity were studied. Peripheral blood lymphocytes (PBL) and large granular lymphocytes (LGL, 70–90% purity) were used as effector cells and K562 as targets. Overnight incubation of the effector cells with TGF-β1 resulted in a significant inhibition of NK activity. TGF-β1 did not influence the expression of CD3, CD 16, CD 18 or CD56 antigens on PBL. Combination of TGF-βl with indomethacin gave the same NK-suppressive effect as TGE-β1 alone, showing that the inhibition of NK acuvity by TGF-β1 is not due to an increase in PGE₂ levels. TGF-β did not influence cAMP level in PBL whereas PGE₂ significantly increased it. On the other hand. TGE-β1 and PGE₂ showed an additive inhibitory effect on NK activity. TGF-β1 did not reduce the binding of PBL and LGL to K562. PGE₂ suppressed the binding and TGF-β1 did not influence this suppression. TGF-β1 also suppressed IL-2-induced activation of NK activity and increase of expression of the granule proteins granzyme A and perforin. PGE₂ did not appear to affect granzyme A and perforin contents. The results indicate that TGF-β1 and PGE₂ suppress NK activity by different mechanisms.     

Ovine and Human Placental IgG inhibit Human Natural Killer Cell Cytotoxicity in vitro

To determine the effect of ovine and human placental IgG on human Natural Killer (NK) cell cytotoxicity in vitro placental IgG was eluted at acidic pH and purified by ion exchange and subsequently by affinity chromatography on protein G and protein A sepharose columns. These antibodies were analysed for presense of IgG by immuno-electrophoresis and relative purity determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). The effect of these antibodies on human NK cell cytotoxicity was investigated by slChromium Release Assay using human K562 cells as targets and human peripheral blood lymphocytes as effector cells. Both ovine and human placental IgG inhibited human NK cell cytotoxicity in a dose dependent manner. Placental IgG may down-regulate the cytotoxic effects of NK cells in vivo by competitively excluding the binding of NK cells to their respective targets on the trophoblast. Alternatively, these antibodies may themselves be toxic to NK cells. Either way, the presence of these antibodies on the placental trophoblast may prevent the binding of NK cells and subsequent immunological rejection of the fetal allograft. Also, ovine placental IgG may be functionally similar to its human counterpart and may therefore be suitable as a model for the study of maternal fetal interaction during pregnancy in humans.     

Nitric Oxide Synthase Pathway May Mediate Human Natural Killer Cell Cytotoxicity

The present study provides evidence that the human natural killer (NK) cell effector mechanism causing target cytolysis has a requirement for L-arginine. In a deficient medium (DM) containing only salts, buffer system and glucose, NK cell-mediated cytotoxicity was found to decrease by 70% as compared to that obtained in a complete medium (CM). However, adding L-arginine to such DM could restore the activity of NK cells to the normal level. Many other components of CM, such as serum, glutamine and vitamins did not improve NK cell-mediated killing in DM. When all amino acids except L-arginine were added to DM only a partial recovery of NK cell functional cytolysis was seen. L-arginine enhanced the NK cell activity in a dose-dependent manner. Additionally, the inhibitor of both inducible and constitutive nitric oxide synthase, N-monomethyl-L-arginine (L-NMMA) inhibited NK cytolytic activity in DM supplemented with L-arginine indicating participation of nitric oxide (NO). The results also show that the stimulatory effect of L-arginine on human NK cell-mediated cytotoxicity was accompanied by an increase in NO formation as determined by accumulation of nitrite and citrulline. L-NMMA gave a dose-dependent reduction in NO generation as well. The nitrite and citrulline production dose-dependenlly correlated with not only the concentration of L-arginine in the cultivation medium, but also the enhanced NK cell-mediated cytolysis. Taken together, these findings could define a L-arginine/NO-linked effector mechanism in human NK cells. Nitrite and citrulline were not formed when NK cell-mediated target cell killing took place in a L-arginine-free DM supplemented with additives. Thus, it appears as if human NK cells may cause target cell killing via both NO-dependent and -independent processes.     

Free Oxygen Radicals Are Not Detectable by Chemiluminescence during Human Natural Killer Cell Cytotoxicity

Mononuclear cells isolated from peripheral blood of normal donors produce free oxygen radicals (FR), delectable by chemiluminescence (CL). when interacting with target cells during natural killer (NK) cell lysis. FR-producing cells were found to have monocyte characteristics and gave a positive CL reaction when mixed at low concentration (0.5%) with purified NK cells. No correlation was found between susceptibility to NK cell lysis and capacity to induce CL with different target cell lines. Using high and low molecular FR scavengers, no NK cell inhibition was seen with superoxide dismutase, cytochrome c, and catalase, whereas some inhibition was seen with 4,5-dihydroxy-in-benzcnedisulphonic acid (Tironø) and 2,3-dihydroxybenzoatc. These compounds, however, required higher concentraiions than used for inhibition of CL, suggesting an alternative action of these compounds. Normal levels of NK cell activity were found in two patients with chronic granulomatous disease, who were genetically incapable of producing detectable amounts of FR. As a result, it is concluded that human NK cells do not produce large amounts of FR during killing and that FR are unlikely to be the lytic end product. Nevertheless, neither a low degree of FR formation in NK cells nor a more subtle signal-transmitting role of FR during NK cell triggering can be excluded.     

Natural Killer Cell Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity to Herpes Simplex Virus-Infected Cells in Human Pregnancy

ABSTRACT: Natural killer cell (NKC) cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) represent the ability of human leukocyte effector cells to destroy target cells in the absence and presence of antibody, respectively. Since these immune systems play a pivotal role in the body's primary lines of defense against a variety of pathogens including herpes simplex virus (HSV), a study was undertaken to evaluate the influence of pregnancy on these systems. Eleven uncomplicated gravidas were followed serially through each trimester and compared to 11 nonpregnant female controls. Mononuclear cells were acquired by Ficoll-Hypaque centrifugation of heparinized blood. Chang liver cells infected with HSV-I were utilized as target cells in a ⁵¹Cr release assay. Mean NKC values in the pregnant patients were uniformly lower than in the controls. No similar decreases in ADCC activity were observed in a comparison between the two study populations. These data support previous observations suggesting that pregnancy represents a relatively immunocompromised state. Differences apparently exist between NKC and ADCC effector cell populations with regard to the influence of pregnancy. Although these physiologic alterations in immunoregulation may help support the fetoplacental allograph, detrimental conditions may exist regarding susceptibility to various pathogens such as HSV.     

D1 and D2 receptor-mediated dopaminergic modulation of visual responses in cat dorsal lateral geniculate nucleus

The modulatory effects of dopamine (DA) on the visual responses of relay cells of the dorsal aspect of cat lateral geniculate nucleus (dLGN) were tested using local micro-iontophoretic application of DA and application of the receptor-specific agonists SKF38393 (SKF, D₁/D₅) and quinpirole (QUIN, D₂/D₃/D₄) in the anaesthetized alcuronium-treated cat. The effects of DA and QUIN were clearly dose-dependent: small amounts caused a weak and transient facilitation of visual activity (10–30 % increase) preferentially in Y-type relay cells, which changed to a moderate reduction of visual responses when the dose was increased (50 %, maximal 70 %). The effect of SKF was mainly suppressive and increased with the amount of drug applied (up to 90 % reduction). The selective antagonists SCH23390 (SCH, D₁) and sulpiride (SULP, D₂) reduced the effects of co-applied DA agonists. We found little evidence for a specific dopaminergic modulation of the surround inhibition (stimulus-driven lateral inhibition) although DA slightly facilitated the transmission of weak signals (small stimuli). Nevertheless, some dopaminergic effects seem to be mediated via inhibitory interneurons regulating the strength of sustained or recurrent inhibition. Application of DA agonists during blockade of GABA_A receptors indicates a direct suppression of relay cells via D₁ receptors, an excitation of relay cells via D₂ receptors and - with increasing amounts of D₂ agonist - probably also an excitation of inhibitory interneurons, which results in an indirect inhibition of dLGN relay cells (predominantly of the X-type). The results are discussed in relation to the impairment of visual functions in Parkinson's disease.     

Inhibition of Natural Killer Cell Activity by Antigen-Antibody Complexes

The natural killer (NK) cell activity of human peripheral blood mononuclear cells was found to be inhibited by precipitated tetanus toxoid anti-tetanus toxoid complexes (Te/aTe) as well as soluble Te/aTe. Preincubation of the immune complexes with protein A decreased the inhibition of NK cell activity. When mononuclear cells were preincubated with interferon (IF) or interleukin 2 (Il-2) before incubation with Te/aTe, the immune complex-induced inhibition was decreased, while IF or Il-2 added after incubation with the immune complexes had no effect. Using NK cell-enriched suspensions in a single cell agarose assay, the immune complexes were shown to inhibit NK cell activity by inhibiting the formation of effector/target cell conjugates.     

Regulation of Natural Killer Cytotoxicity by Escherichia coli-Derived Human Interferon Gamma

The abilities of Escherichia coli-derived human interferon gamma (IFN-gamma) and E. coli-derived human interferon-alpha A (IFN-alpha A) or -alpha 2 (IFN-alpha 2) to augment natural killer (NK) cytotoxicity were compared. When low concentrations (less than 10 antiviral units/ml) of interferons were used, and equal numbers of antiviral units of E. coli-derived IFN-gamma and E. coli-derived IFN-alpha A or IFN-alpha 2 were compared for their ability to augment NK, E. coli-derived IFN-gamma was found to be more active in augmenting NK against the K562 targets, than E. coli-derived IFN-alpha A or IFN-alpha 2. Antiviral units in these experiments were determined by the standard cytopathic effect assay using vesicular stomatitis virus (VSV)-challenged human fibroblasts, trisomic for chromosome 21. However, when these interferons were compared on a weight basis (ng/ml) or on a molar basis, their ability to augment NK against the K562 targets was comparable. These differences in the relative abilities of these interferons (when their concentrations were expressed in antiviral units/ml) to augment NK, were due to an approximately 100-fold difference in their specific activities (antiviral units per mg of interferon). These were 1.8 X 10(6) units/mg for E. coli-derived IFN-gamma, 2.0 X 10(8) units/mg for E. coli-derived IFN-alpha A, and 1.8 X 10(8) units/mg for E. coli-derived IFN-alpha 2. At concentrations higher than 10 units/ml, all these interferons showed a similar ability to augment NK. Studies on the kinetics of the augmentation revealed that in vitro treatment with E. coli-derived IFN-gamma for several hours was necessary for augmentation of NK against targets from haemopoietic human tumour cell lines (K562, Daudi). In contrast, alpha interferons were able to augment NK after treatment in vitro for significantly shorter periods (30 min or less with certain donors). Augmentation of NK cytotoxicity of human peripheral blood mononuclear leucocytes by E. coli-derived IFN-gamma was not accompanied by the induction of interleukin 2 (IL-2) production, suggesting that IL-2 is not involved in the augmentation of NK by IFN-gamma. A monoclonal antibody specific for human IFN-gamma blocked augmentation of NK by E. coli-derived IFN-gamma and natural IFN-gamma, but not by E. coli-derived IFN-alpha A or staphylococcal enterotoxin A (SEA).(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of Human Natural Killer Cell Activity by Platelet-Derived Growth Factor (PDGF)

We have previously reported that platelet-derived growth factor (PDGF) substantially inhibits human natural killer (NK) cell cytotoxicity, and that NK cells possess high-affinity surface binding sites for the PDGF-AB isoform. In this communication, we present direct evidence for the presence of A-type (alpha) PDGF receptors on human NK cells by demonstrating that human NK cells have approximately 150,000 high-affinity, surface binding sites for recombinant (r)PDGF-AA and approximately 300,000 high-affinity, surface binding sites for rPDGF-BB. This was determined by the competitive binding of 125I-labelled rPDGF-AA or 125I-labelled rPDGF-BB and homologous unlabelled rPDGF-AA or rPDGF-BB to FACS-sorted, CD16+ lymphoid (NK) cells, and Scatchard analysis of these data. In addition, we also demonstrate that the various isoforms of PDGF have differential effects on NK-cell cytotoxicity. Physiological quantities (100 ng/ml) of rPDGF-BB homodimers, highly purified PDGF-AB heterodimers from outdated platelets, and rPDGF-AB heterodimers substantially inhibited NK-cell cytotoxicity in both a dose- and time-dependent manner. In contrast, pretreatment of NK cells with equivalent nanogram amounts of rPDGF-AA homodimers resulted in a significantly weaker inhibitory effect on NK-cell cytotoxicity as compared with the PDGF-BB and PDGF-AB isoforms. The implications of these findings are discussed.     

Role of prostaglandin D2 and E2 terminal synthases in chronic rhinosinusitis

BACKGROUND: Prostaglandin (PG)D(2) and E(2), two major cyclooxygenase (COX) products, are generated by PGD(2) synthase (PGDS) and PGE(2) synthase (PGES), respectively, and appear to mediate airway inflammation. OBJECTIVE: We sought to determine the role of PGDS and PGES in the pathophysiology of chronic rhinosinusitis (CRS). METHODS: The study examined the expression of PGDS and PGES in nasal polyps of 22 CRS patients. As controls, uncinate process mucosae were obtained from 12 CRS patients not having nasal polyps and five subjects without sinusitis. Immunohistochemistry and quantitative real-time PCR were used to evaluate the expression. RESULTS: Both PGDS and PGES were detected in nasal polyps by immunohistochemistry. Significantly greater levels of PGDS mRNA and lesser levels of PGES mRNA were observed in the nasal polyps as compared with uncinate process mucosae, and an inverse correlation between PGDS and PGES expression was observed. Levels of PGDS mRNA in nasal polyps were positively correlated with degree of infiltration by EG2+ eosinophils, whereas the levels of PGES were inversely correlated. Significantly increased levels of PGDS and conversely decreased levels of PGES were observed in asthmatics as compared with non-asthmatics. In addition, PGDS and PGES levels were positively and inversely correlated with the radiological severity of sinusitis, respectively. CONCLUSIONS: These results suggest that PGDS and PGES display an opposite and important role in the pathophysiology of CRS such as polyp formation, and more specifically, a biased expression of these synthases might contribute to the development of CRS by affecting eosinophilic inflammation.     

Effect of ASTA-Z 7575 (INN Maphosphamide) on Human Lymphokine-Activated Killer Cell Induction

Abstract

Recent studies combining chemotherapeutic agents with various biological response modifiers for the treatment of cancer have shown promising results. Cyclophosphamide (Cy) is the most widely used alkylating agent and a major constituent of combination chemotherapy regimens for many neoplastic diseases. It has been reported that Cy is a cytotoxic drug, which becomes immunosuppressive at higher doses. A synthetic metabolite of Cy, ASTA-Z, has recently been produced. ASTA-Z is more active and stable by itself and does not need to be metabolically converted to an active compound. the combined effect of Cy and interleukin-2 (IL-2) on the induction of lymphokine-activated killer (LAK) cells is not known. Therefore, we decided to investigate the effect of ASTA-Z on the induction and function of LAK. the coculture of peripheral blood mononuclear cells (PBMC) with various concentrations of ASTA-Z (0, 10^?6 10^?5, 10^?4 and 10^?3 dilution) and IL-2 (50 U/ml) for 4 days produced significant suppression of cytotoxicity and lytic ability of the LAK cells against NK-sensitive (K562) and NK-resistant (M14) tumor cell lines. the lower doses of ASTA-Z did not affect the generation of LAK cells, its cytotoxicity and lytic ability of ASTA-Z against both NK-sensitive and NK-resistant tumor cell lines. Furthermore, the ASTA-Z produced dose-dependent suppression of the proliferative response of LAK cells. the significant therapeutic benefit in the cancer patient may be achieved by the low dose regimen of Cy and IL-2 because it has no deleterious effect on the induction and function of LAK cells.     

Target Cell Lysis by Natural Killer Cells is Influenced by β2-Microglobulin Expression

Natural killer (NK) cells form part of the vertebrate defence against viruses and tumours, but show only limited specificity. The molecule(s) recognized by NK cells on target cells are at present unknown. Major histocompatibility complex (MHC) class I antigen concentration on target cells is inversely correlated with NK cell lysis. Here we show that MHC class I-unassociated beta 2-microglobulin (beta 2-m) expression is involved in NK cell-target cell interaction. Two human MHC class I negative cell lines, Daudi and K562, are differentially susceptible to NK cell lysis. Daudi cells are beta 2-m-negative and resistant to NK lysis, K562 are beta 2-m-positive and highly susceptible to lysis by NK cells. Interferon (IFN) treatment augments beta 2-m expression and NK lysis of K562, but not in Daudi cells. NK cell lysis of K562, but not YAC-1 cells, can be inhibited by monoclonal anti-human beta 2-m antibody. Furthermore, susceptibility of mouse embryo fibroblasts (MEF) to NK lysis can be increased by infection with recombinant vaccinia virus expressing the human beta 2-m gene.     

Effect of ASTA-Z 7575 (INN Maphosphamide) on Human Lymphokine-Activated Killer Cell Induction

Recent studies combining chemotherapeutic agents with various biological response modifiers for the treatment of cancer have shown promising results. Cyclophosphamide (Cy) is the most widely used alkylating agent and a major constituent of combination chemotherapy regimens for many neoplastic diseases. It has been reported that Cy is a cytotoxic drug, which becomes immunosuppressive at higher doses. A synthetic metabolite of Cy, ASTA-Z, has recently been produced. ASTA-Z is more active and stable by itself and does not need to be metabolically converted to an active compound. the combined effect of Cy and interleukin-2 (IL-2) on the induction of lymphokine-activated killer (LAK) cells is not known. Therefore, we decided to investigate the effect of ASTA-Z on the induction and function of LAK. the coculture of peripheral blood mononuclear cells (PBMC) with various concentrations of ASTA-Z (0, 10^-6 10^-5, 10^-4 and 10^-3 dilution) and IL-2 (50 U/ml) for 4 days produced significant suppression of cytotoxicity and lytic ability of the LAK cells against NK-sensitive (K562) and NK-resistant (M14) tumor cell lines. the lower doses of ASTA-Z did not affect the generation of LAK cells, its cytotoxicity and lytic ability of ASTA-Z against both NK-sensitive and NK-resistant tumor cell lines. Furthermore, the ASTA-Z produced dose-dependent suppression of the proliferative response of LAK cells. the significant therapeutic benefit in the cancer patient may be achieved by the low dose regimen of Cy and IL-2 because it has no deleterious effect on the induction and function of LAK cells.     

Effector Cell Sensitivity to Sugar Moieties. I. Inhibition of Human Natural Killer Cell Activity by Monosaccharides

Human natural killer (NK) cells recognize multiple target antigens. The ligands (antigens) involved in the effector-target cell interaction have not been extensively identified. In the present study, assays of NK activity in the presence of a panel of monosaccharides demonstrated inhibition of cytolysis in a dose-response fashion. We propose that NK cell activity involves the recognition of carbohydrate structures on target cells via receptors on the effector cell surface.     

Reproducibility of leukotriene D4 inhalation challenge in asthmatics

We have studied the reproducibility of a bronchial leukotriene (LT) provocation test in asthmatics, and the effect of prior treatment with an oral leukotriene D4/E4 antagonist (SR 2640) on LTD4-induced bronchoconstriction in nine asthmatics in a double-blind placebo-controlled randomized cross-over trial. The reproducibility of the bronchial leukotriene provocation test was high. For a specific patient, the replication variance is 0.2303, and the standard deviation is thus 0.4799, corresponding to 48%, i.e. one halving of the dose or half doubling of the dose. SR 2640 antagonised LTD4 induced bronchoconstriction causing a mean shift of 48% to the right of the dose-response curve as compared with placebo (95% confidence interval being 11-137%). This study demonstrates that bronchial LTD4 provocation test is a safe and reproducible method in asthmatics, and that the method can be used to detect LT-antagonism; furthermore that SR 2640 is a weak LTD4-antagonist in asthmatics.     

A comparison of the biochemical properties of the human diaphorase (DIA3) isozymes determined by the common alleles DIA13, DIA23 and DIA33

(1) Various buffer systems for the starch gel electrophoresis of human diaphorase isozymes have been explored. Electrophoresis in a Tris/Borate system at pH 8.6 which includes 70 micron NADH in the gel and cathodal electrode buffers, provides good resolution of the six DIA3 phenotypes previously resolved by isoelectric focusing. (2) The variant genes DIA13, DIA23 and DIA33 occur with frequencies of about 0.76, 0.23 and 0.01 respectively in the English population. (3) The isozymes determined by the least common gene, DIA33, are markedly different from the isozymes determined by DIA13 and DIA23 in their relatively low heat stability, high affinity for Blue Sepharose and slow anodal electrophoretic mobility in buffer systems containing borate. The DIA3 1 and DIA3 2 isozymes are similar to one another in these characteristics.