Intracellular zinc homeostasis in leukocyte subsets is regulated by different expression of zinc exporters ZnT-1 to ZnT-9

Intracellular zinc homeostasis is strictly regulated by zinc binding proteins and zinc transporters. In the present study, we quantified in a first global view the expression of all characterized human zinc exporters (hZnT-1-9) in different leukocyte subsets in response to zinc supplementation and depletion and analyzed their influence on alterations in the intracellular zinc concentration. We found that hZnT-1 is the most regulated zinc exporter. Furthermore, we discovered that hZnT-4 is localized in the plasma membrane similar to hZnT-1. hZnT-4 is most highly expressed in Molt-4, up-regulated after treatment with PHA and is responsible for the measured decrease of intracellular zinc content after high zinc exposure. In addition, we found that hZnT-5, hZnT-6, and hZnT-7 in Raji as well as hZnT-6 and hZnT-7 in THP-1 are up-regulated in response to cellular zinc depletion. Those zinc exporters are all localized in the Golgi network, and this type of regulation explains the observed zinc increase in both cell types after up-regulation of their expression during zinc deficiency and, subsequently, high zinc exposure. Furthermore, we detected, for the first time, the expression of hZnT-8 in peripheral blood lymphocytes, which varied strongly between individuals. While hZnT-2 was not detectable, hZnT-3 and hZnT-9 were expressed at low levels. Further on, the amount of expression was higher in primary cells than in cell lines. These data provide insight into the regulation of intracellular zinc homeostasis in cells of the immune system and may explain the variable effects of zinc deficiency on different leukocyte subsets.

Effect of zinc supplementation on the immune status of healthy older individuals aged 55–70 years : the ZENITH Study

Aging is associated with alterations in the immune system, effects which may be exacerbated by inadequate zinc (Zn) status. We examined the relationship between Zn status and markers of immunity and the effect of supplementation with 15 mg or 30 mg Zn/d for 6 months on immune status in healthy individuals. Zn status was assessed by dietary intake and biochemical indices. Immune status was assessed by multiple flow cytometric methods. At baseline, Zn concentration was positively associated with lymphocyte subpopulation counts and T-lymphocyte activation. Zn supplementation of 30 mg/d significantly lowered B-lymphocyte count, albeit at month 3 only. Lower doses of Zn (15 mg Zn/d) significantly increased the ratio of CD4 to CD8 T lymphocytes at month 6. Overall, these findings suggest that total Zn intake (diet plus supplementation) of up to 40 mg Zn/d do not have significant long-term effects on immune status in apparently healthy persons aged 55–70 years.

Whole blood analysis of phagocytosis, apoptosis, cytokine production, and leukocyte subsets in healthy older men and women: the ZENITH study

Few studies to date have examined age-related changes in markers of immune status in healthy older individuals. The immune status of 93 healthy individuals aged 55–70 years was assessed by two- and three-color flow cytometry and biochemical analysis. There were significant age effects (p ≤.05) on monocyte phagocytic activity and cluster of differentiation (CD) 3/human leukocyte antigen-D-related (HLA-DR) late-activated T lymphocytes (% expression). There was a significant (p ≤ 0.1) Age x Sex interaction in absolute counts (x 10⁹/L) of CD3/CD8 total cytotoxic T lymphocytes (CTL), the CD4 T- helper to CD8 CTL ratio, the CD3/CD4/CD45RA naïve T helper to CD3/CD4/CD45RO memory T helper lymphocyte ratio, and interleukin (IL)-1ß (% expression) by activated monocytes. The study shows that alterations in markers of immune status occur between 55 and 70 years, and provides reference values for the lymphocyte measures in healthy men and postmenopausal women in this age group. The study further highlights the need for sex-specific reference ranges for such markers.

A pseudosymmetric cell adhesion regulatory domain in the β7 tail of the integrin α4β7 that interacts with focal adhesion kinase and src

The β7 integrins α4β7 and Eβ7 play key roles in forming the gut-associated lymphoid tissue, and contribute to chronic inflammation. The α4β7 integrin-mediated adhesion of activated lymphocytes is largely due to a transient increase in avidity from ligand-induced clustering of α4β7 at the cell-surface. Here, we report that L and D enantiomers of a cell-permeable peptide YDRREY encompassing residues 735-740 of the cytoplasmic tail of the β7 subunit inhibit the adhesion of T cells to β7 integrin ligands. The YDRREY peptide abrogated mucosal addressin cell adhesion molecule-1-induced clustering of α4β7 on the surface of activated T cells. A mutated form of the YDRREY peptide carrying either single or double conservative mutations at Tyr⁷³⁵Phe and Tyr⁷⁴⁰Phe was unable to inhibit T cell adhesion, suggesting that both tandem tyrosines are critical for activity. The YDRREY peptide was bound and phosphorylated by focal adhesion kinase and src, which may serve to sequester cytoskeletal proteins to the cytoplasmic domain of 4β7. The quasi-palindromic sequence YDRREY within the β7 cytoplasmic tail constitutes a cell adhesion regulatory domain that modulates the interaction of β7-expressing leukocytes with their endothelial and epithelial ligands. Cell-permeable peptidomimetics based on this motif have utility as anti-inflammatory reagents for the treatment of chronic inflammatory disease.

Studies of the human lymphocyte P2Z receptor and its activation of phospholipase D

Extracellular adenosine 5′-triphosphate (ATP) is an agonist for the P2Z receptor of human leukaemic lymphocytes and opens a Ca ²⁺-selective ion channel, which also conducts Ba²⁺, Sr²⁺ and the small fluorescent dye, ethidium⁺. A wide range of receptor agonists, many of which raise cytosolic [Ca²⁺] activate phospholipase D (PLD). In the present study, it was shown that both ATP and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) stimulated PLD activity in a concentration-dependent manner, and the inhibitory effects of suramin, oxidised ATP, extracellular Na⁺ and Mg²⁺ suggested that the effect of these agonists is mediated by P2Z receptors. The role of divalent cations in ATP-stimulated PLD activity was investigated. Several agonists (eg ATP, thapsigargin, ionomycin) stimulated a rise in cytosolic [Ca²⁺] in human lymphocytes, but only ATP and ionomycin stimulated PLD activity. When Ca²⁺ influx was prevented by EGTA, the majority of ATP-stimulated and all of ionomycin-stimulated PLD activity was inhibited. Preloading cells with the Ca²⁺ chelator, BAPTA, reduced cytosolic [Ca²⁺] and, paradoxically, ATP-stimulated PLD activity was potentiated. ATP-stimulated PLD activity was supported by both Ba²⁺ and Sr²⁺ when they were substituted for extracellular Ca²⁺. Furthermore, both ATP-stimulated PLD activity and ATP-stimulated ¹³³Ba²⁺ influx showed a linear dependence on extracellular [Ba²⁺]. Thus it was concluded that ATP stimulated PLD activity in direct proportion to the influx of divalent cations through the P2Z ion channel and this PLD activity was insensitive to changes in bulk cytosolic [Ca²⁺]. The calmodulin (Ca²⁺/CaM) inhibitor, trifluoperazine (TFP) inhibited ionomycin- and ATP-stimulated PLD activity and ATP-stimulated apoptosis, but had no effect on PLD activity already activated by ATP. However, TFP inhibited ATP-stimulated Ca²⁺, Ba²⁺ and ethidium⁺ fluxes, at concentrations below those which inhibit Ca²⁺/CaM, suggesting that TFP inhibits the P2Z receptor. Similarly, the isoquinolinesulphonamide, KN-62, a selective inhibitor of Ca²⁺/CaM-dependent protein kinase II (CaMKII), also prevented ATP-stimulated apoptosis, but had no effect on pre-activated PLD. In addition, KN-62, and an analogue, KN-04, which has no effect on CaMKII, potently inhibited ATP-stimulated Ba²⁺ influx (IC₅₀ 12.7 ± 1.5 and 17.3 ± 2.7 nM, respectively), ATP-stimulated ethidium⁺ uptake (IC₅₀ 13.1 ± 2.6 and 37.2 ± 8.9 nM, respectively), ATP-stimulated phospholipase D activity (50% inhibition 5.9 ± 1.2 and 9.7 ± 2.8 nM, respectively) and ATP-induced shedding of the surface adhesion molecule, L-selectin (IC₅₀ 31.5 ± 4.5 and 78.7 ± 10.8 nM, respectively). They did not inhibit phorbol ester- or ionomycin-stimulated PLD activity or phorbol ester-induced L-selectin shedding. Neither KN-62 nor KN-04 (both 500 nM) have any effect on UTP-stimulated Ca²⁺ transients in fura-2-loaded human neutrophils, a response which is mediated by the P2Y₂ receptor, neither did they inhibit ATP-stimulated contractile responses mediated by the P2X₁ receptor of guinea pig urinary bladder. Thus, KN-62 and KN-04 are almost equipotent as P2Z inhibitors with IC₅₀s in the nanomolar, indicating that their actions cannot be due to CaMKII inhibition, but rather that they are potent and direct inhibitors of the P2Z receptor. Extracellular ATP-induced shedding of L-selectin from lymphocytes into the medium is a Ca²⁺-independent response. L-selectin is either cleaved by a metalloproteinase or a PLD with specificity for glycosylphosphatidylinositol (GPI). The novel hydroxamic acid-based zinc chelator, Ro-31-9790 blocks ATP-induced L-selectin shedding, but was without effect on ATP-induced Ba²⁺ influx or ATP-stimulated PLD activity. Furthermore, another zinc chelator, 1,10-phenanthroline, an inhibitor of a GPI-PLD, potentiated rather than inhibited ATP-stimulated PLD activity, suggesting that ATP-induced L-selectin shedding and ATP-stimulated PLD activity are independent of each other. Although extracellular ATP is the natural ligand for the lymphocyte P2Z receptor, it is less potent than BzATP in stimulating Ba²⁺ influx. Concentration-response curves for BzATP- and ATP-stimulated ethidium⁺ influx gave EC₅₀s 15.4 ± 1.4 µM and 85.6 ± 8.8 µM, respectively. The maximal response to ATP was only 69.8 ± 1.9% of that for BzATP. Hill coefficients were 3.17 ± 0.24 and 2.09 ± 0.45 for BzATP and ATP respectively, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z-operated ion channel. A rank order of agonist potency of BzATP > ATP = 2MeSATP > ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. When ATP (300 -1000 µM) was added simultaneously with 30 µM BzATP (EC₉₀), it reduced both ethidium⁺ and Ba²⁺ fluxes by 30 - 40% relative to values observed with BzATP alone. KN-62, previously shown to be a specific inhibitor of the lymphocyte P2Z receptor, was a less potent antagonist of BzATP-induced fluxes than ATP, when maximal concentrations of both agonists (50 and 500 µM respectively) were used. However, when BzATP (18 µM) was used at a concentration equiactive with a maximally effective ATP concentration, KN-62 showed the same inhibitory potency for both agonists. The ecto-ATPase antagonist, ARL-67156, inhibited both ATP- and BzATP-stimulated Ba²⁺ influx, suggesting that the lower efficacy of ATP compared with BzATP was not due to preferential hydrolysis of ATP. Thus, the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a full agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes. Both ATP- and BzATP-stimulated PLD activity were significantly inhibited (P < 0.05) when cells were suspended in iso-osmotic choline Cl medium. Choline⁺ was found to be a permeant for the P2Z ion channel, since ATP induced a large uptake of [¹⁴C]choline⁺ (60 to 150 µmol/ml intracellular water) during a 5 min incubation, which remained in the cells for several hours, and ATP was used to load cells with these levels of choline⁺. Intracellular choline⁺ inhibited ATP-, BzATP-, PMA- and ionomycin-stimulated PLD activity. Brief exposure of lymphocytes to ATP increased the subsequent basal rate of ethidium⁺ uptake, and this was prevented by intracellular choline⁺. It is proposed that P2Z-mediated Ca²⁺ influx in lymphocytes activates PLD leading to significantly changes of the phospholipid composition of the plasma membrane, which subsequently produces a permeability lesion, which in turn contributes to cell death.

Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in ApoE-deficient mice

In the study, we investigate whether the expressions of heat shock protein (hsp)60 (a potential autoantigen) and the stress-inducible form of cytoprotector hsp70 are correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E–deficient (apoE^-/-) mice. The apoE^-/- mouse model is advantageous because the stress-inducible form of hsp70 is not constitutively expressed in mice, unlike primates; hence, tissues under stress can be clearly defined. Both mammalian hsps were detected newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia at lesion-prone sites of 3-week-old apoE^-/- mice. In 8- and 20-week-old mice, they were strongly and heterogeneously expressed in early to advanced fibrofatty plaques, with levels correlating with lesion severity. Expression was markedly downregulated in advanced collagenous, acellular, calcified plaques of 40- and 69-week-old mice and was absent in control aortas of normocholesterolemic wild-type (apoE^+/+) mice. Western blot analysis of tissue homogenates confirmed the temporal expression of the hsps. Double immunostaining revealed that both hsps were expressed by lesional endothelial cells, macrophages, smooth muscle cells, and CD3⁺ T lymphocytes. This study provides evidence that hsp60 and hsp70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis, suggesting that few cells escape the toxic environment of the atherosclerotic plaque.

Anti-inflammatory immunotherapy for multiple sclerosis/experimental autoimmune encephalomyelitis (EAE) disease

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory diseases of the central nervous system (CNS) characterized by localized areas with demyelination. Disease is believed to be an autoimmune disorder mediated by activated immune cells such as T- and B-lymphocytes and macrophages/microglia. Lymphocytes are primed in the peripheral tissues by antigens, and clonally expanded cells infiltrate the CNS. They produce large amounts of inflammatory cytokines, nitric oxide (NO) that lead to demyelination and axonal degeneration. Although several studies have shown that oligodendrocytes (OLGs), the myelin-forming glial cells in the CNS, are sensitive to cell death stimuli, such as cytotoxic cytokines, anti-myelin antibodies, NO, and oxidative stress, in vitro, the mechanisms underlying injury to the OLGs in MS/EAE remain unclear. The central role of glutamate receptors in mediating excitotoxic neuronal death in stroke, epilepsy, trauma and MS has been well established. Glutamate is the major excitatory amino acid transmitter within the CNS and it's signaling is mediated by a number of postsynaptic ionotropic and metabotropic receptors. Inflammation can be blocked with anti-cell adhesion molecules MAb, simultaneously protected oligodendrocytes and neurons against glutamate-mediated damage with the AMPA/kainate antagonist NBQX, and the NMDA receptor antagonist GPE, could thus be effective therapies for multiple sclerosis.

Mouse B7-H3 induces antitumor immunity

Members of the B7 family costimulate the proliferation of lymphocytes during the initiation and maintenance of antigen-specific humoral and cell-mediated immune responses. While B7-1 and -2 are restricted to lymphoid tissues, and activate naïve T cells, recently identified members including B7-H2 and -H3 are widely expressed on nonlymphoid tissues, and regulate effector lymphocytes in the periphery. B7-H3 has properties that suggested it may display antitumor activity, including the ability to stimulate Th1 and cytotoxic T-cell responses. Here, we test this notion by determining whether intratumoral injection of an expression plasmid encoding a newly described mouse homologue of B7-H3 is able to eradicate EL-4 lymphomas. Intratumoral injection of a mouse B7-H3 pcDNA3 expression plasmid led to complete regression of 50% tumors, or otherwise significantly slowed tumor growth. Mice whose tumors completely regressed resisted a challenge with parental tumor cells, indicating systemic immunity had been generated. B7-H3-mediated antitumor immunity was mediated by CD8(+) T and NK cells, with no apparent contribution from CD4(+) T cells. In summary, the results indicate that B7-H3 interactions may play a role in regulating cell-mediated immune responses against cancer, and that B7-H3 is a potential therapeutic tool.

Rhotekin 2, a novel GTPase effector involved in resistance to apoptosis

This thesis describes the discovery, characterization, and function of a new protein, Rhotekin 2. It is a signaling protein with a significant role in resistance to cell death in human lymphocytes and therefore may be a potential new target in future treatments of immune diseases such a HIV and leukemia.

Location rather than CD62L phenotype is critical in the early establishment of influenza-specific T cell memory

The rapid recall of influenza virus-specific CD8+ T cell effector function is protective, although our understanding of T cell memory remains incomplete. Recent debate has focused particularly on the CD62L lymph node homing receptor. The present analysis shows that although functional memory can be established from both CD62Lhi and CD62Llo CD8+ T cell subsets soon after initial encounter between naive precursors and antigen, the optimal precursors are CD8+CD44hiCD25lo immune lymphocytes isolated from draining lymph nodes on day 3.5 after influenza virus infection. Analysis of primed T cells at different times after challenge indicates that the capacity to transfer memory is diminished at the peak of the primary cytotoxic T lymphocyte response, challenging speculations that the transition to memory first requires full differentiation to effector status. It seems that location rather than CD62Lhi/lo phenotype may be the more profitable focus for further dissection of the early establishment of T cell memory.

Influenza A virus-specific CD8.sup.+ T-cell responses : from induction to function

Seasonal influenza virus infection is a leading cause of illness and mortality in young children and the elderly each year. Current influenza vaccines generate protective antibody responses; however, these must be given annually to provide protection against serologically distinct viruses. By contrast, CD8.sup.+ T cells are capable of recognizing conserved antigenic determinants within the influenza virion and, as such, may provide protection against a number of variant strains of the virus. CD8.sup.+ T cells play a critical key role in controlling and resolving influenza virus infections via the production of cytokines and cytolytic mediators. This article focuses on the induction of the influenza-specific CD8.sup.+ T-cell response and how these cells acquire and maintain effector function after induction. Moreover, we discuss how cytotoxic T-lymphocyte function correlates with protection following vaccination.

Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton

Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4+ T cells. We now show that HIV-1 latency can be established in resting CD4+ T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4+ T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4+ T cells during normal chemokine-directed recirculation of CD4+ T cells between blood and tissue.

Labeling of multiple HIV-1 proteins with the biarsenical-tetracysteine system

Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.

Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway

Background and objective: Natural killer (NK) and natural killer T (NKT)-like cells represent a small but important proportion of effector lymphocytes that we have previously shown to be major sources of pro-inflammatory cytokines and granzymes. We hypothesized that these cells would be increased in the airway in chronic obstructive pulmonary disease (COPD), accompanied by reduced expression of the inhibitory receptor CD94 (Kp43) and increased expression of cytotoxic mediators granzyme B and perforin.
Methods: We measured NK and NKT-like cells and their expression of CD94 in the blood of COPD patients (n = 71; 30 current and 41 ex-smokers), smokers (16) and healthy controls (25), and bronchoalveolar lavage fluid (BALF) from a cohort of subjects (19 controls, 12 smokers, 33 COPD). Activation was assessed by measuring CD69 in blood and the cytotoxic potential of NK cells by measuring granzymes A and B, and using a cytotoxicity assay in blood and BALF.
Results: In blood in COPD, there were no significant changes in the proportion of NK or NKT-like cells or expression of granzyme A or NK cytotoxic potential versus controls. There was, however, increased expression of granzyme B and decreased expression of CD94 by both cell types versus controls. The proportion of NK and NKT-like cells were increased in BALF in COPD, associated with increased NK cytotoxicity, increased expression of granzyme B and decreased expression of the inhibitory receptor CD94 by both cell types.
Conclusions: Treatment strategies that target NK and NKT-like cells, their cytotoxicity and production of inflammatory mediators in the airway may improve COPD morbidity.

Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies.