Regulation of apoptosis by type III interferons

Abstract. Objective: Two types of interferons (IFNs), type I (IFN‐α/β) and type III (IFN‐λs), utilize distinct receptor complexes to induce similar signalling and biological activities, including recently demonstrated for IFN‐λs antitumour activity. However, ability of type III IFNs to regulate cell population growth remains largely uncharacterized.Materials and methods: Intact and modified human colorectal adenocarcinoma HT29 cells were used to study regulation of apoptosis by IFN‐λs. Results and Conclusions: We report that the IFN‐λR1 chain of the type III IFN receptor complex possesses an intrinsic ability to trigger apoptosis in cells. Signalling induced through the intracellular domain of IFN‐λR1 resulted in G₁/G₀ phase cell cycle arrest, phosphatidylserine surfacing and chromosomal DNA fragmentation. Caspase‐3, caspase‐8 and caspase‐9 were activated; however, pancaspase inhibitor Z‐VAD‐FMK did not prevent apoptosis. In addition, the extent of apoptosis correlated with the level of receptor expression and was associated with prolonged IFN‐λ signalling. We also demonstrated that the ability to trigger apoptosis is a unique intrinsic function of all IFN receptors. However, more robust apoptosis was induced by signalling through type III IFN receptor than through type I or type II (IFN‐γ) receptors, suggesting higher cytotoxic potential of type III IFNs. In addition, we observed that IFN‐γ treatment sensitized HT29 cells to IFN‐λ‐mediated apoptosis. These results provide evidence that type III IFNs, alone or in combination with other stimuli, have the potential to induce apoptosis.     

Degradation of monomeric and fibrillar type III collagens by human skin collagenase. Kinetic constants using different animal substrates

Human skin collagenase activity was examined against type III collagens, in both soluble and fibrillar form, from different animal species. In either form, human, dog, and cat type III were degraded 10- to 30-fold faster than was that from guinea pig and nearly 100-fold more readily than chick type III. These differences in susceptibility were mirrored by essentially identical differences in the rate of trypsin cleavage of the same substrates. Human, dog, and cat type III were cleaved most rapidly by trypsin, guinea pig III more slowly, and chick III was completely resistant to the serine protease. Arrhenius plots, relating enzyme activity to temperature, revealed differences in the various type III substrates consistent with their collagenase and trypsin susceptibilities. Human, dog, and cat type III collagens yielded nonlinear plots, with accompanying activation energies which decreased at temperatures above 26 degrees C; guinea pig type III displayed a plot which deviated only slightly from linearity while the plot for chick type III was completely linear. These data strongly suggest that type III collagens display substantial variability in the stability of the helix at or near the collagenase cleavage site. The susceptibility of these type III substrates as reconstituted fibrils was also examined. The relative rates of degradation of these substrates by collagenase, and by trypsin, were the same as those observed in solution. The absolute rates of degradation of collagen in fibrillar form, however, were massively lower than predicted by extrapolation from solution values. This reduction in rate is even greater for type III than for type I collagens. Thus, whereas in solution type III substrates are cleaved much faster than type I collagens, in fibrillar form these differences are less than 2-fold. These data, together with values for activation energies and deuterium isotope effects on type III fibrillar substrates, reinforce the concept that helical integrity near the collagenase cleavage site is a major specifier of the rate of collagenase activity. Furthermore, the data suggest that the exclusion of water accompanying the tight packing of monomers into fibrils presents a major energy barrier to collagenase activity, which is particularly large for type III collagen.     

Regulation of natural killer cytotoxicity by 1,25-dihydroxyvitamin D3

The steroid hormone 1 alpha, 25-dihydroxyvitamin D3, calcitriol, is crucial in calcium homeostasis. Calcium plays a central role in T, B, and NK cell functions, and calcitriol is a known inhibitor of T cell proliferation and immunoglobulin production. We have analyzed here the immunoregulatory effects of calcitriol on NK cell function. We show that calcitriol specifically specifically inhibits, in a time- and dose-dependent fashion, the generation of cytotoxic activity from cultured CD16+ peripheral blood NK cells. It also suppresses, at similar molar concentrations (1-10 nM), interleukin 2 (IL-2) production by PHA-activated peripheral blood lymphocytes. Calcitriol does not interfere with the cytotoxic function of NK cells, whether fresh or generated in vitro, placing the inhibition at the level of NK cell activation. Interestingly enough, exogenous IL-2 can completely reverse the suppressive effect. These findings suggest that modulation of NK cell activation by control of the internal level of IL-2 may reflect an additional paracrine calcitriol-dependent circuit with immunoregulatory consequences.     

Mechanism and regulation of natural cytotoxicity. Minireview on cancer research

Natural killer (NK) cells comprise a heterogeneous population of effector cells functionally and phenotypically distinct from B cells and mature antigen-sensitive T cells, with the capacity to spontaneously lyse target cells of widely different tissue provenance in a genetically unrestricted fashion. As such they have been widely implicated in immunosurveillance against neoplastic and virus-infected cells, as well as in the homeostasis of haematopoietic differentiation and regulation of immune function. In common with cytotoxic T cells, the lytic mechanism may be resolved into several discrete stages. Target cell recognition appears to involve several chemical entities, while susceptibility is also influenced by a multiplicity of factors operative at post-recognition stages of the lytic process. NK activity is subject to both positive and negative regulation. The potentiating effects of interferons and interleukin-2, products of activated T cells, indicate a possible pathway by which adaptive immune responses may augment natural cytotoxicity under local physiological conditions. Negative regulation is mediated by certain prostaglandins and a variety of cell types including macrophages, granulocytes and thymocytes as well as subsets of peripheral blood lymphocytes.     

Role of serotonin in the regulation of human natural killer cell cytotoxicity

Addition of serotonin to mixtures of target cells and natural killer (NK)-enriched human mononuclear cells (MNC) in a 4-hr 51Cr-release assay strongly augmented NK cell cytotoxicity (NKCC) vs K562, Chang, or Molt-4 target cells. The effect was dose dependent at serotonin concentrations of 10(-4) to 10(-7) M, expressed at several effector to target cell ratios, and required the presence of accessory monocytes. A 5-HT1-specific receptor agonist, 8-OH-DPAT, mimicked the enhancing properties of serotonin with similar potency. Equimolar concentrations of the mixed 5-HT1/5-HT2 receptor antagonist cyproheptadine, but not the 5-HT2-specific antagonist ketanserin, completely blocked the serotonin-induced NKCC enhancement. Monocyte/NK cell mixtures incubated with serotonin for 1 hr produced a soluble factor that could enhance the cytotoxicity of autologous, NK-enriched cells depleted of monocytes, which did not respond to serotonin alone. The factor displayed no IFN or IL 2 activity as judged by the lack of antiviral activity and inability to support the growth of an IL 2-dependent cell line. In the presence of monocytes, serotonin (10(-5) M) was considerably more effective than human IFN-alpha or IFN-gamma at optimal concentrations and was about equally effective as IL 2 at a final concentration of 50 U/ml in a short-term NK assay. The potency and efficacy for serotonin were similar to that earlier reported for histamine in monocyte-containing effector cells. The NKCC-enhancing effect of serotonin was additive to that induced by IFN-alpha, IFN-gamma, or IL 2, but not to histamine. The presented data suggest an earlier unrecognized, serotonin receptor-mediated regulation of human NK cells.     

Regulation of antibody-dependent cellular cytotoxicity by IgG intrinsic and apparent affinity for target antigen

Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However, the factors that regulate the magnitude of ADCC are incompletely understood. In this study, we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10(-7) to 10(-11) M, and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10(-10) M (the lowest affinity variant) to almost 10(-11) M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting, a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs.     

Interaction of S-sulfonated human IgG with human complement and its components.

S-sulfonated human IgG (S-sIgG) was prepared by treating IgG with sodium sulfite and sodium tetrathionate. The treatment resulted in the selective cleavage of interchain disulfide bonds of the IgG to give S-sulfonate groups. Complement fixing activities of aggregated S-sIgG and the immune complex formed with the S-sIgG antibody were very weak. S-sIgG at a high dose reduced the activity of the first complement component (C1) in normal human serum without any reduction of other complement components activites, but S-alkylated IgG at the same dose did not. Loss of C1 activity was not caused by either S-sulfonated myeloma proteins (IgA and IgE) or urea-treated S-sIgG, in which both inter- and intra-chain disulfide bonds were cleaved. These results suggest that the selective reduction of C1 by S-sIgG is due to a conformational change of the immunoglobulin.     

Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation

NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.     

Regulation of human natural killing by levamisole

Summary The effect of levamisole on human natural killing (NK) has been studied. In short-term chromium release assays, levamisole at a concentration of 10^–3 M was inhibitory to NK when present in the assays. Pretreatment of NK effector cells and K562 target cells with levamisole separately indicated that the effect was on effector cell activity and was not due to any change in target cell susceptibility. Inactivation of the effector cells required greater than 4 h pretreatment with levamisole if NK activity was subsequently tested in the absence of the drug. Pretreatment with levamisole for up to 19 h had no effect on the lymphocyte proliferative response to phytohemagglutinin (PHA). NK activity of drug-inactivated effector cells recovered after further incubation in levamisole-free medium. Levamisole at 10^–4 M or less had no effect on NK either by pretreatment or by its presence in the NK assays.     

Augmentation of human natural cell-mediated cytotoxicity by recombinant human interleukin 2

Human peripheral blood mononuclear cells (PBMC) demonstrated increased natural cell-mediated cytotoxicity (NCMC) activity after only 5 min of exposure to purified recombinant human IL 2 or interferon (IFN)-gamma. The mechanism of NCMC augmentation by treatment with IL 2 is not entirely dependent on IFN-gamma production because: a) IL 2 was found to augment NCMC activity at levels which did not induce detectable IFN-gamma; b) IL 2 required only 5 min of exposure to PBMC to augment NCMC activity, whereas 3 hr of contact were required to demonstrate detectable IFN-gamma levels; c) the levels of NCMC enhancement by treatment with IL 2 exceeded the amount of NCMC enhancement that could be due to IFN alone; d) anti-recombinant IFN-gamma, which totally eliminated the augmentation of NCMC enhancement by IFN-gamma, only partially reduced the augmentation of NCMC activity by IL 2; and e) combination treatment of PBMC with IL 2 and IFN-gamma resulted in a synergistic enhancement of NCMC. The results strongly support the conclusion that augmentation of NCMC by IL 2 and IFN-gamma involve overlapping mechanisms.     

Interaction between proteolytic strains of Lactococcus lactis influenced by different types of proteinase during growth in milk.

The influence of the type of cell envelope-located proteinase (PI versus PIII) on the associative growth of Lactococcus lactis in milk was studied. Two genetically engineered strains, differing only by the type of proteinase, were first used as a model study. An interaction occurred during the second exponential growth phase of the mixed culture and resulted in a decrease in growth rate of the PI-type proteinase strain, whereas that of the PIII-type proteinase strain remained unaffected. The reduction in proteolytic activity of the PI-type proteinase strain (presumably resulting from an inhibition of the synthesis of the enzyme) due to the peptides released by the PIII-type proteinase was found to be partly responsible for this interaction. Extension of the study to wild-type proteinase-positive L. lactis strains showed a systematic imbalance of the mixture of the two strains in favor of the PIII-type proteinase strain.     

Regulation of monomeric dynein activity by ATP and ADP concentrations.

Axonemal dyneins are force-generating ATPases that produce ciliary and flagellar movement. A dynein has large heavy chain(s) in which there are multiple (4-6) ATP-binding consensus sequences (P-loops) as well as intermediate and light chains, constituting a very large complex. We purified a monomeric form of dynein (dynein-a) that has at least three light chains from 14S dyneins of Tetrahymena thermophila and characterized it. In in vitro motility assays, dynein-a rotated microtubules around their longitudinal axis as well as translocated them with their plus-ends leading. ATPase activity at 1 mM ATP was doubled in the presence of a low level of ADP (> or = 20 microM). Both ATPase activity and translocational velocities in the presence of ADP (> or = 20 microM) fit the Michaelis-Menten equation well. However, in the absence of ADP (< 0.1 microM), neither of the activities followed the Michaelis-Menten-type kinetics, probably due to the effect of two ATP-binding sites. Our results also indicate that dynein-a has an ATP-binding site that is very sensitive to ADP and affects ATP hydrolysis at the catalytic site. This study shows that a monomeric form of a dynein molecule regulates its activity by direct binding of ATP and ADP to itself, and thus the dynein molecule has an intramolecular regulating system.     

Comparison between human immunodeficiency virus positive and negative patients with tuberculosis in Southern Brazil.

The objective of this study is to determine the different characteristics of human immunodeficiency virus (HIV) positive and negative patients treated for tuberculosis (TBC) in a tertiary hospital in Southern Brazil. We conducted a retrospective cohort study over a 5-year period, from January 1992 through December 1996. We reviewed medical charts of patients from our institution who received TBC treatment. We reviewed 167 medical charts of patients with confirmed TBC. HIV positivity was detected in 74 patients. There were statistically significant difference between HIV positive and negative patients in sex and age. HIV-infected patients showed significantly more signs of bacteremia than HIV-negative patients. Extra-pulmonary TBC was present respectively in 13 (17.6%) and 21 (22.6%) HIV positive and negative patients. There was a significant difference between chest radiograph presentation in HIV positive and negative patients. There were significantly lower hematocrit, hemoglobin, leukocyte and lymphocyte levels in HIV-positive compared to HIV-negative patients. Outcome was significantly different in the two groups with a death rate of 36.5% and 10.8% in HIV-positive and in HIV-negative patients. The difference between HIV positive and negative patients may have importance in the diagnosis, management and prognosis of patients with TBC.