Human purified protein derivative-specific CD4+ T cells use both CD95-dependent and CD95-independent cytolytic mechanisms

CTL, both CD4+ and CD8+, are essential in the eradication of intracellular pathogens. Data generated using murine T cells have suggested a critical role for CD95 (Fas, Apo-1) in CD4+ T cell-induced apoptosis of target cells. In contrast, CD8+ CTL predominantly use the perforin/granzyme lytic pathway. At present little is known about the mechanism of CD4+ CTL lytic function during intracellular infection in humans. We have used human CD4+ T cells specific for purified protein derivative (PPD) of Mycobacterium tuberculosis to explore whether CD95 is the dominant cytolytic mechanism. PPD-reactive CD4+ clones efficiently lysed Ag-pulsed autologous monocytes, adherent macrophages, and EBV-transformed B cells. Addition of an antagonistic CD95 Ab had a minimal effect on cytolysis, whereas addition of MgEGTA to block perforin/granzyme resulted in complete inhibition of killing. In contrast, lysis of activated peripheral blood B cells could be partially blocked with the antagonistic CD95 Ab. Supporting these observations, monocytes, macrophages, and EBV-transformed B cells were not lysed by an agonistic CD95 Ab. Activated B cells were readily lysed by the agonistic CD95 Ab. T cell clones triggered through the TCR with anti-CD3 were capable of lysing the CD95-sensitive Jurkat T cell line in a CD95-dependent manner, but were also able to release granzymes. We conclude that human CD4+ T cells are capable of lysing PPD-pulsed targets using both perforin/granzyme and CD95 pathways. The contribution of CD95 is strictly dependent on target cell susceptibility to CD95-mediated killing.     

Granzymes D, E, F, and G are regulated through pregnancy and by IL-2 and IL-15 in granulated metrial gland cells

Granulated metrial gland (GMG) cells are NK cells that proliferate and differentiate within the murine uterus during pregnancy. They have been predicted to play important roles in nurturing the embryo, normal placentation, and uterine tissue remodeling. GMG cell differentiation is manifested by the accumulation of the cytolytic mediators, perforin, granzyme A, and granzyme B, within cytoplasmic granules. The signaling mechanisms required for GMG cell differentiation are largely unknown, although recent in vitro assays have implicated IL-15 in these events. In this report, we demonstrate that granzymes D, E, F, and G (granzymes D-G) are also expressed in GMG cells but at a later stage in pregnancy when compared with granzyme A expression. Whereas granzyme A is expressed in early to mid-gestation, the expression of granzymes D-G peak in mid- to late gestation. In addition, we show that the expression patterns of IL-2Rbeta and the IL-2Rgamma mRNAs overlap with that of granzyme D-G mRNAs in the pregnant uterus. Finally, we demonstrate that granzymes D-G are up-regulated by IL-2 and IL-15 in primary cultures containing GMG cells. Taken together, these results suggest that IL-2 and/or IL-15 may regulate GMG cell differentiation in vivo, and that granzymes D-G may have different functions than granzyme A during pregnancy.     

BCL-2 blocks perforin-induced nuclear translocation of granzymes concomitant with protection against the nuclear events of apoptosis

Cytolytic granule-mediated target cell killing is effected in part through the synergistic action of the membrane-acting protein perforin and serine proteases such as granzymes (Gr) A and B. In this study, we examine the subcellular distribution of granzymes in the presence of perforin and the induction of apoptosis in mouse FDC-P1 myeloid and YAC-1 lymphoma cells that express the proto-oncogene bcl2. Using confocal laser scanning microscopy to visualize and quantitate subcellular transport of fluoresceinated granzyme, we find that granzyme entry into the cytoplasm in the absence of perforin is not impaired in the bcl2-expressing lines. However, perforin-dependent enhancement of granzyme cellular uptake and, importantly, granzyme redistribution to the nucleus were strongly inhibited in the bcl2-expressing lines, concomitant with greatly increased resistance to granzyme/perforin-induced cell death. DNA fragmentation induced by granzyme/perforin was severely reduced in the bcl2-expressing lines, implying that prevention of granzyme nuclear translocation blocks the nuclear events of apoptosis. The kinetics of GrB nuclear uptake and induction of apoptosis were faster than for GrA, whereas YAC-1 cells showed greater resistance to granzyme nuclear uptake and apoptosis than FDC-P1 cells. In all cases, granzyme nuclear accumulation in the presence of perforin correlated precisely with ensuing apoptosis. All results supported the idea that GrA and GrB share a common, specific nuclear targeting pathway that contributes significantly to the nuclear changes of apoptosis.     

Extracellular granzymes A and B in humans: detection of native species during CTL responses in vitro and in vivo

Activated CTLs and NK cells induce apoptosis via multiple mechanisms, including that termed granule exocytosis. The latter pathway consists of vectorial secretion of perforin and a family of granule-associated serine proteases (granzymes) to the target cell. To establish whether granzymes are released extracellularly during cytolytic reactions in vivo, ELISAs that measure the native enzymes were developed and were found to specifically detect granzyme A (GrA) and granzyme B (GrB) at picogram concentrations. Low levels of GrA and GrB were present in plasma of healthy individuals (GrA, 33.5 pg/ml (median); GrB, 11.5 pg/ml (median)), whereas significantly higher levels were present in patients with ongoing CTL response, i.e., patients suffering from infections by EBV or HIV type 1. Markedly elevated levels were also noted in synovial fluid of patients with active rheumatoid arthritis. The measurement of soluble granzymes should be useful to assess clinical disorders associated with activated CTL and NK cells. Furthermore, these results suggest that granzymes mediate biologic effects beyond their described role in apoptotic cell death.     

Interrelationship between ectoparasites and wild rodents from Tijucas do Sul, state of Paraná, Brazil

NK cells kill sensitive targets via exocytosis of cytoplasmic granules containing membrane damaging perforins and DNA damaging granzymes. Therefore, the target cell can either die by necrosis or by apoptosis. A third, non-secretory, mechanism of killing mediated by Fas-FasL interaction can be used by activated NK cells to kill Fas+ targets. Here, we have studied the modulation exerted by two NK active cytokines, IL-2 and IL-12, on the apoptosis-inducing activity of NK cells in NK-sensitive Fas- K562 and NK-insensitive Fas+ Raji cells. Our results show that apoptosis is preferentially induced at low target:effector ratios. Resting NK cells virtually do not induce apoptosis in target cells while IL-2 stimulation endows NK cells with the capacity of inducing apoptosis and IL-12 further enhances this effect against both targets. Finally, we demonstrate that cytokine-stimulated NK cells use both granzyme and Fas-FasL pathways of apoptosis induction.     

Genetic controls of meiotic recombination and somatic DNA metabolism in Drosophila melanogaster

Recombination-defective meiotic mutants and mutagen-sensitive mutants of D. melanogaster have been examined for their effects on meiotic chromosome behavior, sensitivity to killing by mutagens, somatic chromosome integrity, and DNA repair processes. Several loci have been identified that specify functions that are necessary for both meiotic recombination and DNA repair processes, whereas mutants at combination and DNA repair processes, whereas mutants at other loci appear to be defective in only one pathway of DNA processing.     

Phase variation of lipopolysaccharide directs interconversion of invasive and immuno-resistant phenotypes of Neisseria gonorrhoeae

Phase variation of Neisseria gonorrhoeae lipopolysaccharide (LPS) controls both bacterial entry into human mucosal cells, and bacterial susceptibility to killing by antibodies and complement. The basis for this function is a differential sialylation of the variable oligosaccharide moiety of the LPS. LPS variants that incorporate low amounts of sialic acid enter human mucosal epithelial cells very efficiently, but are susceptible to complement-mediated killing. Phase transition to a highly sialylated LPS phenotype results in equally adhesive but entry deficient bacteria which, however, resist killing by antibodies and complement because of dysfunctional complement activation. Phase variation of N. gonorrhoeae LPS thus functions as an adaptive mechanism enabling bacterial translocation across the mucosal barrier, and, at a later stage of infection, escape from the host immune defence.     

How do cytotoxic lymphocytes kill their targets?

In situ killing of tumor cells using suicide gene transfer to generate death by a non-apoptotic pathway was associated with high immunogenicity and induction of heat shock protein (hsp) expression. In contrast, a syngeneic colorectal tumor line, CMT93, killed predominantly by apoptosis, showed low levels of hsp expression and less immunogenicity. When apoptosis was inhibited in CMT93 cells by overexpression of bcl-2, hsp was also induced. Furthermore, when cDNA encoding hsp70 was stably transfected into B16 and CMT93 cells, its expression significantly enhanced the immunogenicity of both tumors. Increased levels of hsp, induced by non-apoptotic cell killing, may provide an immunostimulatory signal in vivo which helps break tolerance to tumor antigens. These findings have important implications for the development of novel anti-cancer therapies aimed at promoting patients' immune responses to their own tumors.     

Alternative pathway of complement in ruminants: role in infection

Besides being initiated by antigen-antibody complexes, the classical pathway also can be activated by two innate mechanisms: (1) A family of proteins called collectins, which resemble C1q, bypass the activation of C1q. (2) Acute phase proteins, belonging to the family of pentraxins, activate the classical pathway by binding to C1q. The term 'alternative' complement pathway is a misnomer. This system is a primary primitive immune mechanism. It is phylogenetically older than the classical pathway. Contrary to the classical pathway, it does not require development of a specific immune response before getting into action. It acts within minutes after the microorganism has entered the body. The alternative pathway is continually activated at a low controlled rate but amplified by the surface of intruding microorganisms. It has the capacity to distinguish between self and non-self. Many nonpathogenic microorganisms are killed by the alternative pathway of complement. Pathogens have developed evasion mechanisms to escape the killing effect of this pathway. The kinetics of the activation of the alternative pathway of ruminants differs from that of mouse and man. The difference might be mediated by conglutinin.     

Zinc and metallothioneins on cellular immune effectiveness during liver regeneration in young and old mice

Partial hepatectomy in young mice (pHx) induces thymic atrophy, disregulation of thymocytes subsets and a strong accumulation of zinc in thymic tissue after 1-2 days of liver regeneration. Zinc is relevant for good immune functioning. Restoration of zinc into both the thymus and thymocytes subsets in the late period of liver regeneration is observed in young pHx mice. These findings have suggested a link between the thymus and the liver influencing T-cell functions and involving zinc. This kind of link could be relevant in aging because thymic involution, negative crude zinc balance and crippled immune functions are constant events. The preminence of a liver extrathymic T-cell pathway after pHx or during aging has been suggested. Thus the study of pHx in young and old mice may offer a good model to better understand the role played both by thymic involution and by liver extrathymic T-cell pathway and the role of zinc in these physiological processes during aging. Young pHx mice after 1-2 days of liver regeneration show: reduced thymic endocrine activity, increment of double negative (DN) thymocytes subsets, impairment of peripheral immune efficiency (PHA, NK activity and IL-2) and negative crude zinc balance, which are all restored in the late period of liver regeneration. By contrast the thymic and peripheral immune defects and the negative crude zinc balance, already present in old sham mice, are not modified during liver regeneration in old pHx mice. Circulating leukocytes and lymphocytes are not significantly modified both in young and old pHx mice as compared to respective sham controls. Zinc may also be crucial for extrathymic T-cells pathway, being preminent in aging, rather than in young age, due to its metallothioneins (MT) binding capacity. MT are significantly increased in young pHx and in aging inducing a low zinc-free quota for thymic and peripheral immune efficiency in young pHx mice, and for extrathymic T-cell pathway, in old age. Thus low zinc bioavailability, due to MT, may play a pivotal role, not only for thymocytes but also for liver extrathymic T-cell pathway.     

Age-dependent alterations of human recombinant GM-CSF effects on human granulocytes

The granulocyte-macrophage colony stimulating factor (GM-CSF) is an important in vivo regulator of granulopoiesis and neutrophil functions. It is well-known that the immune response and the transmembrane signalling in immune cells change with aging. We wished to elucidate the effects of GM-CSF in itself and in priming the activities of other inflammatory agents on neutrophils of elderly persons. Neutrophils of 20 healthy elderly (aged 60-90 years) and 20 healthy young (aged 20-25 years) subjects were studied for superoxide anion production, intracellular free calcium mobilization, antibody dependent cellular cytotoxicity (ADCC) and intracellular killing activities. It was found that GM-CSF is unable to prime neutrophils of elderly subjects to the action of FMLP, metenkephalin or opsonized zymosan. By the use of Pertussis toxin and H7 it was demonstrated that a different signal transduction pathway in neutrophils of elderly subjects is activated by GM-CSF or FMLP if compared to that of young subjects. These results suggest that the lack of priming could contribute to the greater susceptibility of the elderly to infections and that the change of the signal transduction mechanism in neutrophils of elderly subjects might partly explain this phenomenon.     

Distinct T cell receptor signaling requirements for perforin- or FasL-mediated cytotoxicity

A diverse array of signals are generated in a cytotoxic T lymphocyte (CTL) after the T cell receptor (TCR) engages the class I major histocompatibility complex (MHC) peptide complex. These signals result in a multitude of CTL effector functions, including cellular cytotoxicity, cell surface receptor expression, and cytokine secretion. We have examined signaling through the TCR in a wild type CD8+, MHC-restricted, antigen-specific CTL clone, 14-7, and its interleukin 2-dependent variant clone 14-7FD. We report here that 14-7FD is unable to kill via the perforin mechanism of killing, yet is able to kill via the Fas ligand/Fas mechanism and secrete interferon-gamma in an antigen-specific manner. 14-7FD has cytolytic granules that contain perforin and serine esterases, which are secreted after phorbol ester and Ca2+ ionophore treatment. Lastly, to investigate which TCR signaling requirements were operational in 14-7FD, we examined TCR-triggered intracellular Ca2+ mobilization in the two clones. After TCR engagement, 14-7FD failed to mobilize intracellular Ca2+, which may be the cause for its inability to trigger the perforin/granule exocytosis mechanism of killing. These results indicate that the signal transduction events that trigger perforin killing and the signaling requirements to induce FasL expression are distinct. We hypothesize that these two distinct TCR signal transduction requirements allow for separate activation of these two mechanisms of killing relating to their role in eradication of infected cells or regulation of immune responses.     

Rapid purification of cationic granule proteases: application to human granzymes

This report describes a simple scheme for the simultaneous purification of cationic human granzymes A, B, and 3 from human interleukin 2 (IL-2)-activated lymphocytes. The process, which requires approximately 8 h, includes: (1) cell cavitation, (2) two centrifugation steps, (3) four granule solubilization steps, and (4) cation-exchange chromatography. Granule solubilization consists of three extractions with a hypotonic buffer (25 mM NaCl) that contained Triton X-100 followed by a final extraction in hypertonic detergent-free buffer (390 mM NaCl). We recovered approximately 35% of the trypsin-like (tryptase) activities mediated by granzymes A and 3, respectively, and approximately 25% of the asp-ase activity of granzyme B. The granzymes were identified after elution from the Mono S column by Western blot with a polyclonal antibody that reacts with a conserved amino acid sequence (9-16) of lymphoid/myeloid serine proteases. By amino-terminal sequencing, eluted granzyme A and B were indeed homogeneous. Granzyme 3, although highly enriched, appears to be contaminated with an uncharacterized granzyme. Although we have developed this scheme to rapidly isolate the granzymes, the procedure should assist the purification of secretory granule-associated cationic proteins that reside in neutrophils and mast cells as well as other cells that possess secretory function.     

Two signaling mechanisms for activation of alphaM beta2 avidity in polymorphonuclear neutrophils

Circulating polymorphonuclear neutrophils (PMN) are quiescent, nonadherent cells that rapidly activate at sites of inflammation, where they develop the capacity to perform a repertoire of functions that are essential for host defense. Induction of integrin-mediated adhesion, which requires an increase in integrin avidity, is critical for the development of these effector functions. Although a variety of stimuli can activate integrins in PMN, the signaling cascades involved are unclear. Phosphatidylinositol (PI) 3-kinase has been implicated in integrin activation in a variety of cells, including PMN. In this work, we have examined activation of the PMN integrin alphaM beta2, assessing both adhesion and generation of the epitope recognized by the activation-specific antibody CBRM1/5. We have found that PI 3-kinase has a role in activation of alphaM beta2 by immune complexes, but we have found no role for it in alphaM beta2 activation by ligands for trimeric G protein-coupled receptors, including formylmethionylleucylphenylalanine (fMLP), interleukin-8, and C5a. Cytochalasin D inhibition suggests a role for the actin cytoskeleton in immune complex activation of alphaM beta2, but cytochalasin has no effect on fMLP-induced activation. Similarly, immune complex activation of the Rac/Cdc42-dependent serine/threonine kinase Pak1 is blocked by PI 3-kinase inhibitors, but fMLP-induced activation is not. These results demonstrate that two signaling pathways exist in PMN for activation of alphaM beta2. One, induced by FcgammaR ligation, is PI 3-kinase-dependent and requires the actin cytoskeleton. The second, initiated by G protein-linked receptors, is PI 3-kinase-independent and cytochalasin-insensitive. Pak1 may be in a final common pathway leading to activation of alphaM beta2.     

A second serine protease associated with mannan-binding lectin that activates complement

The complement system comprises a complex array of enzymes and non-enzymatic proteins that is essential for the operation of the innate as well as the adaptive immune defence. The complement system can be activated in three ways: by the classical pathway which is initiated by antibody-antigen complexes, by the alternative pathway initiated by certain structures on microbial surfaces, and by an antibody-independent pathway that is initiated by the binding of mannan-binding lectin (MBL; first described as mannan-binding protein) to carbohydrates. MBL is structurally related to the complement C1 subcomponent, C1q, and seems to activate the complement system through an associated serine protease known as MASP (ref. 4) or p100 (ref. 5), which is similar to C1r and C1s of the classical pathway. MBL binds to specific carbohydrate structures found on the surface of a range of microorganisms, including bacteria, yeasts, parasitic protozoa and viruses, and exhibits antibacterial activity through killing mediated by the terminal, lytic complement components or by promoting phagocytosis. The level of MBL in plasma is genetically determined, and deficiency is associated with frequent infections in childhood, and possibly also in adults (for review, see ref. 6). We have now identified a new MBL-associated serine protease (MASP-2) which shows a striking homology with the previously reported MASP (MASP-1) and the two C1q-associated serine proteases C1r and C1s. Thus complement activation through MBL, like the classical pathway, involves two serine proteases and may antedate the development of the specific immune system of vertebrates.     

A low molar ratio of retinol binding protein to transthyretin indicates vitamin A deficiency during inflammation: studies in rats and a posterior analysis of vitamin A-supplemented children with measles

Natural killer (NK) cells have been implicated in early immune responses against certain viruses, including cytomegalovirus (CMV). CMV causes downregulation of class I major histocompatibility complex (MHC) expression in infected cells; however, it has been proposed that a class I MHC homolog encoded by CMV, UL18, may act as a surrogate ligand to prevent NK cell lysis of CMV-infected cells. In this study, we examined the role of UL18 in NK cell recognition and lysis using fibroblasts infected with either wild-type or UL18 knockout CMV virus, and by using cell lines transfected with the UL18 gene. In both systems, the expression of UL18 resulted in the enhanced killing of target cells. We also show that the enhanced killing is due to both UL18-dependent and -independent mechanisms, and that the killer cell inhibitory receptors (KIRs) and CD94/NKG2A inhibitory receptors for MHC class I do not play a role in affecting susceptibility of CMV-infected fibroblasts to NK cell-mediated cytotoxicity.     

A family of bicistronic vectors to enhance both local and systemic antitumor effects of HSVtk or cytokine expression in a murine melanoma model

The herpes simplex virus-thymidine kinase/ganciclovir (HSVtk/GCV) system produces both direct and immune-mediated tumor cell killing. Here, we compare the efficacy of HSVtk/GCV with cytokines, alone and in combination, on the tumorigenicity and immunogenicity of B16 cells. With respect to single gene modifications, only HSVtk/GCV, or high-level interleukin-2 (IL-2) secretion, completely prevented tumor growth, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) generated the best levels of long-term systemic protection. To augment both local killing and immune activation, we constructed bicistronic constructs that express HSVtk and a cytokine within the same cell. Co-expression of HSVtk with IL-2 or GM-CSF enhanced the local antitumor activity of any gene alone. In a tumor-prevention model, HSVtk killing, in an environment preprimed with GM-CSF, generated the best long-term immune protection. However, in a short-term therapy model, continued IL-2 expression was most effective against 3-day established tumors. This probably reflects differences in the activities of IL-2 and GM-CSF in generating short-term, nonspecific immune stimulation compared to long-term immunological memory, respectively. As a prelude to in vivo delivery experiments, we also demonstrated that these bicistronic cassettes can be packaged normally into retroviral (5 x 10(5) virus/ml from pooled populations) and adenoviral vectors (5 x 10(9) virus/ml) and function as predicted within virally infected cells. This family of bicistronic vectors can be used to stimulate synergy between suicide and cytokine genes, overcomes the problems of delivering two genes on separate vectors, and should allow easier preparation of vectors for the delivery of multiple genes to patients' tumor cells.     

The need for power, stress, immune function, and illness among male prisoners.

Previous studies have demonstrated that a strong power motive in college students as assessed by the TAT (nPow), if inhibited and/or stressed, was associated with impaired immune-function reports of more serious illnesses. 133 male prisoners' (mean age 28 yrs) motives were assessed from the TAT, stress and illness from self-report inventories, and immune function from concentrations of immunoglobulin A in saliva (S-IgA). Those high in nPow and in reported stress showed the highest levels of reported illness and the lowest concentrations of S-IgA—significantly different from those high in nPow and low in stress, but not from those simply high in stress. The motive/stress and lowered immune-function connection cannot be attributed to response bias. As expected, high concentrations of S-IgA were associated with reports of fewer upper respiratory infections, supporting the hypothesis that some motive/stress and illness connections may be mediated by impaired immune functions. (18 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)