Hungyi Shau

Modulation of natural killer and lymphokine‐activated killer cell cytotoxicity by lactoferrin

Natural killer (NK) and lymphokine‐activated killer (LAK) cell cytotoxic functions can be strongly augmented by the iron‐carrier protein lactoferrin (LF). LF significantly enhances NK and LAK activities when added at the beginning of NK or LAK cytotoxicity assays. LF is effective in augmenting cytotoxic activities at concentrations as low as 0.75 μg/ml, and higher concentrations of LF induce greater augmentation of NK and LAK. Iron does not appear to be essential for LF to increase NK and LAK, as depleting iron from LF with the chelator deferoxamine does not affect the capacity of LF to increase cytotoxicity. LF is known to have RNase enzymatic activity, and LF enhancement of NK and LAK can be blocked by RNA. However, LFs from two different sources with over 100‐fold difference in RNase activity are equally effective in enhancing NK and LAK. Furthermore, purified non‐LF RNase does not modulate NK or LAK activity and DNA is as effective as RNA in blocking LF augmentation of NK or LAK cytotoxicity. Therefore, the RNase activity is unlikely to be responsible for LF enhancement of the cytotoxicities. Newborn infants are known to have low NK activity and NK and LAK cells have been implicated in host defense against microbial infections. Thus, maternal milk‐derived LF may have a role in boosting antimicrobial immunity in the early stages of life. In adults, LF released from neutrophils may enhance NK and LAK functions in the inflammatory process induced by microbial infections.

Cloning and sequence analysis of candidate human natural killer-enhancing factor genes.

A cytosol factor from human red blood cells enhances natural killer (NK) activity. This factor, termed NK-enhancing factor (NKEF), is a protein of 44000 Mrconsisting of two subunits of equal size linked by disulfide bonds. NKEF is expressed in the NK-sensitive erythroleukemic cell line K562. Using an antibody specific for NKEF as a probe for immunoblot screening, we isolated several clones from a λgt11 cDNA library of K562. Additional subcloning and sequencing revealed that the candidate NKEF cDNAs fell into one of two categories of closely related but non-identical genes, referred to as NKEF A and B. They are 88% identical in amino acid sequence and 71% identical in nucleotide sequence. Southern blot analysis suggests that there are two to three NKEF family members in the genome. Analysis of predicted amino acid sequences indicates that both NKEF A and B are cytosol proteins with several phosphorylation sites each, but that they have no glycosylation sites. They are significantly homologous to several other proteins from a wide variety of organisms ranging from prokaryotes to mammals, especially with regard to several well-conserved motifs within the amino acid sequences. The biological functions of these proteins in other species are mostly unknown, but some of them were reported to be induced by oxidative stress. Therefore, as well as for immunoregulation of NK activity. NKEF may be important for cells in coping with oxidative insults.

DIFFERENTIAL EXPRESSION OF PEROXIREDOXIN SUBTYPES IN HUMAN BRAIN CELL TYPES

The peroxiredoxin (Prx) protein is expressed widely in animal tissues and serves an antioxidant function associated with removal of cellular peroxides. We have cloned two Prx genes and observed differential expression of Prx-I and Prx-II (formerly NKEF-A and NKEF-B) in purified rat brain cell cultures (Sarafian et al. [1998] Mol. Chem. Neuropathol. 34:39-51). We have examined regional and cell-type-specific expression of Prx-I and Prx-II in paraffin sections of human brain using immunohistochemical methods. These studies revealed a clear segregation of expression of these two gene products in different brain cell types. In the cerebral cortex, cerebellum, basal ganglia, substantia nigra, and spinal cord, Prx-I was expressed primarily in astrocytes, while Prx-II was expressed exclusively in neurons. Prx-I was also prominently expressed in ependymal cells and subependymal matrix of substantia nigra and basal ganglia. Prx-II was not expressed at uniform density in all neurons. In general, small neurons such as cerebellar granule neurons displayed little or no staining, while large neurons, such as hippocampal pyramidal and Purkinje neurons were heavily stained. The absence of expression of Prx-I in neurons and the selective expression of Prx-II in large neurons suggest that these antioxidant enzymes serve distinct functional roles that may reflect the different functions and biochemical activities of these cell types. Restricted expression of these genes may also contribute to the selective vulnerability of these cells to a wide variety of neuropathologic conditions.

Interferon-α Primed Tumor-Infiltrating Lymphocytes Combined with Interleukin-2 and Interferon-α as Therapy for Metastatic Renal Cell Carcinoma

AbstractMurine models demonstrate therapeutic synergy for the combination of interleukin-2, interferon-α and tumor-infiltrating lymphocytes. We treated 11 patients with metastatic renal cell carcinoma with a novel regimen consisting of in vivo primed tumor-infiltrating lymphocytes, interferon-α and interleukin-2. Patients received interferon-α before radical nephrectomy; in vivo primed tumor-infiltrating lymphocytes were isolated and expanded in vitro. Low dose continuous infusion inter-leukin-2 at a dose of 2 × 106 units per m2 per day was administered for 96 hours during each treatment week and interferon-α was administered as a subcutaneous injection at a dose of 6 × 106 units per m.2 per day on days 1 and 4 of the interleukin-2 infusion. No therapy was given during the last 3 days of a treatment week. One course of therapy consisted of 3 weeks of therapy followed by 3 weeks of rest. Patients were treated until maximal response, disease progression or dose limiting toxicity. A maximum of 6 courses of t...

Endogenous natural killer enhancing factor-B increases cellular resistance to oxidative stresses.

Natural killer-enhancing factor (NKEF) was identified and cloned on the basis of its ability to increase NK cytotoxicity. Two genes, NKEF-A and -B, encode NKEF proteins and sequence analysis presented suggests that each belongs to a highly conserved family of antioxidants. To examine the antioxidant potential of NKEF, we transfected the coding region of NKEF-B cDNA into the human endothelial cell line ECV304. The stable transfectant, B/1, was found to overexpress NKEF-B gene transcript and protein. We subjected B/1 to oxidative stress by either culturing them with glucose oxidase (GO), which continuously generates hydrogen peroxide, or by direct addition of hydrogen peroxide. We found that B/1 cells were more resistant than control cell lines. Resistance to hydrogen peroxide was originally thought to be mediated mainly by catalase and the glutathione cycle. Therefore, we used inhibitors to block the two pathways and found that B/1 cells were more resistant to oxidative stress than control cells when we used inhibitors to preblock either pathway. We also examined the cellular inflammatory responses to oxidized low-density lipoprotein (LDL) and bacterial lipopolysaccharide (LPS) by measuring monocyte adhesion to endothelial cells in vitro and found that B/1 cells were resistant to such responses. Lastly, we found that B/1 cells were more resistant to a novel chemotherapeutic agent CT-2584, which appears to kill tumor cells by stimulating production of reactive oxygen intermediates in mitochondria. These results demonstrate that the NKEF-B is an antioxidant that protects cells from oxidative stress, chemotherapy agents, and inflammation-induced monocyte adhesion. Furthermore, its expression may mediate cellular responses to proinflammatory molecules.

Recombinant natural killer enhancing factor augments natural killer cytotoxicity.

Natural killer enhancing factor (NKEF) was originally identified and studied because of its ability to enhance NK cytotoxicity in vitro. After cloning the two genes responsible for NKEF proteins, NKEF‐A and ‐B, we found that they belong to a newly described and highly conserved antioxidant gene family. We have now produced recombinant proteins of both genes and used them to test for their ability to promote NK cytotoxicity. Although recombinant NKEF (rNKEF)‐A and ‐B have similar levels of antioxidant function, only the reduced form of rNKEF‐A can enhance NK cytotoxicity. These results indicate that both the antioxidant and NK‐enhancing functions of rNKEF‐A and ‐B probably involve the cysteine residues of the proteins but are mediated by separate domains of the molecules. We pretreated both effector cells and target cells to investigate which population was influenced by rNKEF‐A, and determined that the protein must be present during the cytotoxicity assay to enhance the activity. Despite the similarities between NK cytotoxicity and lymphokine‐activated killer (LAK) cytotoxicity, rNKEF‐A is not effective in augmenting LAK cytotoxicity. Therefore, rNKEFs can be useful tools in not only protecting cells from oxidative damage, but also in selectively promoting NK cytotoxicity against certain tumor cells.

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-B-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 (approximately 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 microM 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.

Contrasting antioxidant and cytotoxic effects of peroxiredoxin I and II in PC12 and NIH3T3 cells.

We examined the impact of peroxiredoxin-I (Prx-I) and peroxiredoxin-II (Prx-II) stable transduction on oxidative stress in PC12 neurons and NIH3T3 fibroblasts and found variability depending on cell type and Prx subtype. In PC12 neurons, Prx-II suppressed reactive oxygen species (ROS) generation by 36% (p < 0.01) relative to vector-infected control cells. However, in NIH3T3 fibroblasts, Prx-II overexpression resulted in a 97% (p < 0.01) increase in ROS generation. Prx-I transduction elevated ROS generation in PC12 cells. The effect of Prx-I on PC12 cells was potentiated in the presence of menadione, and suppressed by an inhibitor of nitric oxide synthetase. Prx-II transduction resulted in 25–35% lower levels of glutathione (GSH) in both cell types, while Prx-I transduction increased GSH levels in neurons and decreased GSH and caspase-3 activity in fibroblasts. Prx-I and Prx-II also had differing effects on cell viability. These results suggest that Prx-I and Prx-II can either increase or decrease intracellular oxidative stress depending on cell type or experimental conditions, particularly conditions affecting nitric oxide levels.

IL-6 enhances the cytotoxic activity of thymocyte-derived CD56+ cells

Thymocyte-derived lymphokine-activated killer (LAK) cells were used as a model for the study of the cytokine driven development of cytotoxicity. These cells are devoid of initial cytotoxic activity but upon culture in IL-2 they develop into cytotoxic effectors. The parameters of the response of thymocytes to IL-6 are similar to that of PBL in that IL-6, at concentrations as low as 1 mu/ml, increases cytotoxicity of thymocyte-LAK cells when generated in low doses (25-50 mu/ml) of IL-2. IL-6-enhanced thymocyte-LAK cytotoxicity is observed when tested against both NK-resistant and NK-sensitive tumor cell lines. IL-6 alone does not induce any cytotoxicity from thymocytes nor does IL-6 change the time course of thymocyte-LAK cell generation in IL-2 culture. IL-6 does not affect DNA synthesis, total cell number, proportion of CD56+ cells, or the expression of IL-2R (both P55 and P75 glycoproteins) in IL-2-cultured thymocytes. Instead, IL-6 used to treat mature thymocyte-LAK effector cells for as little as 1 hr prior to 51Cr-release assay increases LAK cytotoxicity. This enhancement is abrogated by pretreatment of effector cells with cycloheximide, suggesting that protein synthesis is required for IL-6 to enhance LAK cell activity. The precursor phenotypes of IL-6-responsive thymocyte-LAK cells are CD3-/CD5-. The effector phenotypes of IL-6-enhanced thymocyte-LAK cells are CD5-/CD56+. Thus, IL-6 depends on synthesis of rapid-turnover proteins to act on mature CD56+/CD5- LAK cells to increase their cytotoxic function.

Expression of the antioxidant geneNKEF in the central nervous system

Free radicals and the oxidative stress they impose can cause serious injury in the nervous system and contribute to pathology associated with a wide variety of degenerative and traumatic disorders. In this study, we examined the expression of an antioxidant defense gene, nkef, in human tissue and isolated populations of rat brain cells using Western and Northern blot analysis. NKEF protein was expressed in human brain, liver, kidney, muscle, and lung. The human endothelial cell line ECV expressed a 25-kDa band in addition to the 22-kDa band normally observed. In the central nervous system, a 22-kDa NKEF band was present in cortical gray and white matter, hippocampus, cerebellum, and spinal cord in roughly similar amounts. Expression of NKEF-A and NKEF-B subtypes was evaluated by Northern analysis of cultured cell types from embryonic rat brain. Astrocyte and microglia expressed both 22- and 25-kDa bands, whereas cortical neurons and oligodendrocytes contained only the 22-kDa protein band. Northern blot analysis of these cell types revealed low levels of NKEF-A message in neurons and oligodendrocytes, and relatively low levels of NKEF-B in microglia. Differential expression of these antioxidant defense genes may contribute to the selective vulnerability of brain cell types to specific kinds of oxidative stress.