Modulation of natural killer-mediated lysis by red blood cells

Natural killer (NK)-mediated cytotoxicity is significantly enhanced in the presence of red blood cells (RBC). The enhancement is dose dependent on the number of RBC present and can be induced by autochthonous, allogeneic, or xenogeneic RBC. The enhancement was demonstrated in cytotoxicity against two different NK-sensitive tumor target cell lines, K562 and U937, and by three different assay systems, chromium release, lactate dehydrogenase release, and inhibition of thymidine incorporation. RBC directly enhance the cytotoxic activity of NKH-1/Leu19+ large granular lymphocyte NK cells. Intact RBC have to be present during the cytotoxicity assay to induce the enhancement, which probably occurs at a postbinding, preprogramming phase. The anti-CD2 antibody Leu5 cannot block the enhancement at a concentration inhibitory to lymphocyte rosetting with sheep RBC, suggesting that the enhancement is not induced by interaction through the CD2 antigen. These results indicate that RBC are a potent modulator of NK cytotoxicity and suggest that in vitro NK studies using purified lymphocytes may not always truly reflect NK activity in the blood stream.     

The role of transferrin in natural killer cell and IL-2-induced cytotoxic cell function

The growth factor transferrin (Tf) enhanced natural killer (NK) cell cytotoxicity. This enhancement was due to direct effects on NK cell function, and Tf treatment of the K562 target cell had no effect on their sensitivity. NK cells were highly enriched in the low-density large granular lymphocyte population (LGL) by Percoll gradient centrifugation. Despite the direct effect of Tf on NK cells, the number of cells expressing receptors for Tf (TfR) in NK-enriched LGL was the same as the NK-cell-depleted high-density small lymphocyte population (SL). All populations, tested without stimulation, had very few TfR+ cells. Interleukin 2 (IL-2) could induce very high NK-like activity in the LGL but not in SL. Similarly, only LGL could be induced by IL-2 to express TfR. In serum-free cultures, only limited NK-like activity could be developed which was greatly enhanced by supplementing with Tf in the cultures. The importance of Tf in NK-like development was confirmed by modulating the expression of TfR in IL-2 containing cultures with mouse monoclonal antibody OKT9 specific for TfR. OKT9 totally abrogated the induction of cytotoxic activity by IL-2 against K562 and NK-resistant target. OKT9 inhibited the induction of cytotoxicity in both lymphocytes containing active NK cells and in those predepleted of active NK cells, indicating that the development of NK-like activity from both precursor populations requires Tf. The inhibition by OKT9 was only during the induction phase. The same antibody had no effect on the cytotoxicity of fresh NK cells or the mature IL-2-induced NK-like cells. Our data therefore do not support the hypothesis of TfR as the NK recognition structure. Instead, these results indicate that Tf is important for the development of NK and NK-like activities.     

Functional studies on the precursors of human lymphokine-activated killer cells

Human peripheral blood lymphocytes cultured for 4 days in the interleukin 2 (IL-2)-containing cell-free supernatant of the MLA144 cell line (MLA144CM) are cytolytic to NK-susceptible and NK-resistant tumor target cells. This lymphokine-activated killer (LAK) activity is dependent on IL-2 as development of LAK activity is inhibited in the presence of a monoclonal antibody (MoAb) reacting with the IL-2 receptor (anti-Tac). Addition of cyclosporin A (CyA) to mixed lymphocyte cultures inhibits the development of allospecific cytotoxic activity and inhibits the development of IL-2 responsiveness. However, development of LAK activity is unaffected by the inclusion of CyA in the cultures, showing that the LAK precursor can be functionally distinguished from the allospecific cytotoxic precursor cell. Development of LAK activity does not require mature NK cells as shown by the generation of LAK activity from NK inactive human thymocytes and lymph node cells. In addition, depletion of NK activity from human PBL does not impair the development of LAK activity.     

Characteristics of interleukin-6-enhanced lymphokine-activated killer cell function

To study the effect of IL-6 on the development of cytotoxic cells, we examined lymphokine-activated killer (LAK) activity generated from human nonadherent PBL. Addition of rIL-6 at the initiation of 5-day PBL cultures significantly increases LAK activity in the presence of low concentrations (between 5 and 25 u/ml) of rIL-2. RIL-6 alone induces no PBL LAK activity but at doses as low as 0.8 u/ml rIL-6 enhances LAK activity with optimal enhancement of LAK at 5.0 u/ml of rIL-6. This enhancement is independent of effects on cells growth as rIL-6 did not affect the cell recovery of PBL cultured in rIL-2. RIL-6-enhanced LAK is mediated by the same type of effector cells as those of LAK from rIL-2 alone with effector cells primarily generated from large granular CD3-negative E rosetting lymphocytes. RIL-6 does not change the time course of LAK development and pretreatment of PBL with rIL-6 has no effect on the PBL response to subsequent rIL-2 induction of LAK. Addition of rIL-6 to LAK cultures 2 hr before the cytotoxicity assay shows equal enhancement as addition at the initiation of the culture. However, rIL-6 requires the presence of both rIL-2 and another factor in the supernatant from LAK cultures in order to enhance LAK. Our results indicate that IL-6 can modulate LAK activity at a very late stage of LAK development, and that the enhancement by IL-6 is dependent on the presence of IL-2 and another soluble factor generated during rIL-2 culture.     

Systematics,genetics, and biogeography of intertidal mites (Acari,Oribatida) from the Andaman Sea and Strait of Malacca

This study demonstrates for the first time the presence of marine‐associated mites in the Andaman Sea and Strait of Malacca and reveals a relatively high diversity of these taxa with six species from two different families: Selenoribatidae and Fortuyniidae. Indopacifica, a new genus of Selenoribatidae, is described from Thailand and Malaysia, with two new species, Indopacifica pantai n. sp. and Indopacifica parva n. sp. The genus is characterized by the unique combination of following characters: lacking lamellar ridges, incomplete dorsosejugal suture, fourteen pairs of notogastral setae, and presence of epimeral foveae. A phylogenetic reconstruction based on 18S ribosomal RNA sequences clearly confirms the distinctness of the new genus Indopacifica and places it close to the genus Rhizophobates. The lack of molecular genetic data of possible relatives impedes a clear assessment, and hence, we emphasize the need for further combined approaches using morphological and molecular genetic sequence data. All species show wide distribution areas within this geographic region suggesting that these taxa are good dispersers despite their minute size and wingless body. Molecular genetic data demonstrate recent gene flow between far distant populations of I. pantai n. sp. from the coasts of Thailand and two islands of Malaysia and hence confirm this assumption. The seasonally changing surface currents within this geographic area may favor hydrochorous dispersal and hence genetic exchange. Nevertheless, morphometric data show a slight trend to morphological divergence among the studied populations, whereas this variation is suggested to be a result of genetic drift but also of habitat differences in one population of Alismobates pseudoreticulatus.     

The Discovery of Single-Nucleotide Polymorphisms—and Inferences about Human Demographic History

A method of historical inference that accounts for ascertainment bias is developed and applied to single-nucleotide polymorphism (SNP) data in humans. The data consist of 84 short fragments of the genome that were selected, from three recent SNP surveys, to contain at least two polymorphisms in their respective ascertainment samples and that were then fully resequenced in 47 globally distributed individuals. Ascertainment bias is the deviation, from what would be observed in a random sample, caused either by discovery of polymorphisms in small samples or by locus selection based on levels or patterns of polymorphism. The three SNP surveys from which the present data were derived differ both in their protocols for ascertainment and in the size of the samples used for discovery. We implemented a Monte Carlo maximum-likelihood method to fit a subdivided-population model that includes a possible change in effective size at some time in the past. Incorrectly assuming that ascertainment bias does not exist causes errors in inference, affecting both estimates of migration rates and historical changes in size. Migration rates are overestimated when ascertainment bias is ignored. However, the direction of error in inferences about changes in effective population size (whether the population is inferred to be shrinking or growing) depends on whether either the numbers of SNPs per fragment or the SNP-allele frequencies are analyzed. We use the abbreviation "SDL," for "SNP-discovered locus," in recognition of the genomic-discovery context of SNPs. When ascertainment bias is modeled fully, both the number of SNPs per SDL and their allele frequencies support a scenario of growth in effective size in the context of a subdivided population. If subdivision is ignored, however, the hypothesis of constant effective population size cannot be rejected. An important conclusion of this work is that, in demographic or other studies, SNP data are useful only to the extent that their ascertainment can be modeled.     

Differential susceptibility to staphylococcal superantigen (SsAg)-induced apoptosis of CD4+ T cells from atopic dermatitis patients and healthy subjects: the inhibitory effect of IL-4 on SsAg-induced apoptosis

This study had two aims: 1) to determine whether there are differences between atopic dermatitis (AD) patients and healthy subjects in staphylococcal superantigen (SsAg)-induced CD4(+) T cell activation, cytokine production, chemokine receptor expression, and apoptosis; and 2) to investigate the effect of IL-4 on SsAg-induced apoptosis. By using immunofluorescence and annexin V staining, we analyzed PBMC with or without staphylococcal enterotoxin B (SEB) stimulation in the presence or absence of rIL-4 or anti-IL-4-neutralizing Abs in 15 healthy subjects and 27 AD patients. We found that SEB preferentially induced production of Th1 cytokine in SEB-reactive (TCRVbeta3(+) or Vbeta12(+) or Vbeta17(+)) CD4(+) T cells from healthy subjects and Th2 cytokine in those from AD patients. SEB induced up-regulation of CXCR3(+) cells in SEB-reactive CD4(+) T cells from healthy subjects and CCR4(+) cells in those from AD patients. SEB-reactive CD4(+) T cells from AD patients were more resistant to SEB-induced apoptosis than those from healthy subjects. There was no significant difference between AD and healthy subjects in SEB-induced activation of CD4(+) T cells. CXCR3(+) CD4(+) T cells were more susceptible to SEB-induced apoptosis than CCR4(+) CD4(+) T cells in healthy subjects. Exogenously added IL-4 inhibited SEB-induced apoptosis of SEB-reactive CD4(+) and CXCR3(+) CD4(+) T cells but not of CCR4(+) CD4(+) T cells in healthy subjects. Inhibition of endogenous IL-4 increased SEB-induced apoptosis of SEB-reactive CD4(+) T cells from AD patients. These results might provide new clues to the mechanism that SsAgs contribute to the persistence and exacerbation of allergic skin inflammation in AD.     

Role of proliferation in LAK cell development

Summary Lymphokine-activated killer (LAK) cell activity may be largely the result of activation and/or expansion of peripheral blood natural killer cells by culture with interleukin-2 (IL-2). We have examined the role of proliferation in LAK cell development by either inhibiting or enhancing the proliferative potential of peripheral blood lymphocytes. Inhibition of proliferation was accomplished using irradiation, mitomycin C, or the iron chelator deferoxamine. For each of these agents, a dose-dependent inhibition of proliferation was observed. At doses of inhibitor which nearly completely blocked thymidine uptake, the development of LAK activity was only partially impaired. The mitogenic lectins phytohemagglutinin (PHA) and concanavalin A (Con A) augmented the proliferative response of peripheral blood lymphocytes to IL-2. However, augmentation by PHA, but not Con A, consistently resulted in a decrease in LAK activity. This inhibition of LAK activity by PHA did not appear to be due to inhibition of the effector cell, nor to preferential expansion of irrelevant cells. These data suggests that not all LAK activity is dependent on proliferation, and that high levels of proliferation in the presence of IL-2 do not necessarily lead to LAK activity.This work was supported in part by U.S. Public Health Service Grant CA-34442 (S. H. G.) and pre-doctoral training grant CA-09120 (F. J. R.)     

Cellular Antioxidant Properties of Human Natural Killer Enhancing Factor B

The protein, NKEF (natural killer enhancing factor), has been identified as a member of an antioxidant family of proteins capable of protecting against protein oxidation in cell-free assay systems. The mechanism of action for this family of proteins appears to involve scavenging or suppressing formation of protein thiyl radicals. In the present study we investigated the antioxidant protective properties of the NKEF-B protein overexpressed in an endothelial cell line (ECV304). Nkef-B-transfected cells displayed significantly lower levels of reactive oxygen species (ROS) compared with control or vector-transfected cells. Tert-Butylhydroperoxide-induced ROS was 15% lower in nkef-8-transfected cells and cytotoxicity was slightly, though not significantly, lower. NKEF-B had no effect on ROS induced by menadione or xanthine plus xanthine oxidase. NKEF-B overexpression resulted in slightly (≈ 10%) lower levels of cellular glutathione (GSH) and had no effect on rate or extent of GSH depletion following either diethylmaleate (DEM) or buthionine sulfoximine (BSO) treatment. Lipid peroxidation, assessed as thiobarbituric acid-reactive substances, was 40% lower in nkef-B-transfected cells compared with vector-only-transfected cells. DEM-induced lipid peroxidation was suppressed by NKEF-B at DEM concentrations of 20 μM to 1 mM. At 10 mM DEM, lipid peroxidation was unaffected by NKEF-B. NKEF-B expression also protected cells against menadione-induced inhibition of [³H]-thymidine uptake. The NKEF-B protein appears most effective in suppressing basal low-level oxidative injury such as that produced during normal metabolism. These results indicate that overexpression of the NKEF-B protein promotes resistance to oxidative stress in this endothelial cell line.