The regulatory role of Dipeptidyl peptidase I on the activation of immune granulocytes.

Dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, required for activation of serine proteases of granulocytes including mast cells (MCs), neutrophils (NPs) and others, which were found in synovial tissue of patients with rheumatoid arthritis (RA). But, the role of DPPI associated with those cells in RA development is unclear. In this study, the collagen-induced-arthritis (CIA) rat-model was employed to investigate the expression and activity levels of DPPI and its association with RA progress. Primary granulocytes were freshly extracted from bone-marrows of normal or CIA rats, human mast cell line LAD-2 and primary neutrophils, human-recombinant-DPPI, DPPI-inhibitor Gly-Phe-CHN2 , LTB4, anti-IgE antibody, calcium ionophore were used to study the regulatory role of DPPI in cell activations. The increased DPPI activities in synovial fluids, serum, and bone-marrow homogenates of CIA rats associated with RA severities progress were observed after injections. MMP2/9 expressions in SFs and bone-marrow were in different patterns. Regular-Blood-Tests have shown the high leveled DPPI activities associated with granulocytes differentiations in-vivo in blood of CIA rats. In-vitro cell models, DPPI up-regulated the proliferation of primary bone-marrow granulocytes of normal rats, but inhibited that of CIA rats. DPPI up-regulated and Gly-Phe-CHN2 down-regulated MCs intracellular DPPI and chymase activities. Gly-Phe-CHN2 also inhibited the LTB4 -activated-NPs and NP-elastase activities. Following stimulation of calcium ionophore, the net-releases of DPPI and ß-hexosaminidase from MCs were increased over a time-course, while Gly-Phe-CHN2 down-regulated MCs and NPs activation. Our findings demonstrate the role of DPPI in regulating MCs and NPs activation, and modulating proteolysis in the process of RA.

Beyond low plasma T3: local thyroid hormone metabolism during inflammation and infection.

Decreased serum thyroid hormone concentrations in severely ill patients were first reported in the 1970s, but the functional meaning of the observed changes in thyroid hormone levels, together known as nonthyroidal illness syndrome (NTIS), remains enigmatic. Although the common view was that NTIS results in overall down-regulation of metabolism in order to save energy, recent work has shown a more complex picture. NTIS comprises marked variation in transcriptional and translational activity of genes involved in thyroid hormone metabolism, ranging from inhibition to activation, dependent on the organ or tissue studied. Illness-induced changes in each of these organs appear to be very different during acute or chronic inflammation, adding an additional level of complexity. Organ- and timing-specific changes in the activity of thyroid hormone deiodinating enzymes (deiodinase types 1, 2, and 3) highlight deiodinases as proactive players in the response to illness, whereas the granulocyte is a novel and potentially important cell type involved in NTIS during bacterial infection. Although acute NTIS can be seen as an adaptive response to support the immune response, NTIS may turn disadvantageous when critical illness enters a chronic phase necessitating prolonged life support. For instance, changes in thyroid hormone metabolism in muscle during critical illness may be relevant for the pathogenesis of myopathy associated with prolonged ventilator dependence. This review focuses on NTIS as a timing-related and organ-specific response to illness, occurring independently from the decrease in serum thyroid hormone levels and potentially relevant for disease progression.

Different methods for testing potential cyclooxygenase-1 and cyclooxygenase-2 inhibitors.

The need for the development of selective agents, which only inhibit the mainly "harmful" cyclooxygenase-2 (COX-2) while leaving physiological COX-1 mostly unaffected, still remains, especially after the recent issues related to cardiovascular toxicity caused by some COX-2 selective agents. Thus there is still a demand for sensitive and rapid methods to assay for COX-2 selective agents. Among several in vitro testing systems the whole blood assay (WBA) is a well-known method to examine non-steroidal anti-inflammatory drugs (NSAIDs) in view of their potency to inhibit COX activity. This assay has some major advantages over enzyme-based or isolated cell assays. Emergence of artifacts due to cell separation steps is kept to a minimum and substances, even in disproportional high concentrations, can be examined outside the body in a physiological environment resembling most closely the in vivo conditions in living humans, i.e., 37 degrees C, homeostasis, presence of all blood compounds and cell-cell interactions remain intact. While COX-1 human whole blood assays are performed within less than 2 h, for established COX-2 assays one still has to allow for an overnight incubation step before gaining the desired plasma. The aim of the assay described in this chapter is to characterize an optimized human whole blood assay (hWBA). We present a simple, fast and reliable method to examine the capacity of NSAIDs at inhibiting COX-2 activity that can be applied for rapid and routine screening purposes.

Clinical and biochemical effects of coenzyme Q(10), vitamin E, and selenium supplementation to psoriasis patients.

OBJECTIVE: The aim of the present study was to evaluate clinical effects of supplementation with antioxidants to patients with severe erythrodermic (EP) and arthropathic (PsA) forms of psoriasis. METHODS: Fifty-eight patients were hospitalized, treated by conventional protocols, and randomly assigned to four groups. Groups EP1 and PsA1 were supplemented with coenzyme Q(10) (ubiquinone acetate, 50 mg/d), vitamin E (natural alpha-tocopherol, 50 mg/d), and selenium (aspartate salt, 48 mug/d) dissolved in soy lecithin for 30-35 d. Groups EP2 and PsA2 (placebo) received soy lecithin. Clinical conditions were assessed by severity parameters. Markers of oxidative stress included superoxide production, copper/zinc-superoxide dismutase, and catalase activities in the circulating granulocytes, in the affected epidermis, and plasma levels of nitrites/nitrates. RESULTS: At baseline patients had an increased superoxide release from granulocytes (10.0 +/- 0.5, 2.9 +/- 0.2, and 1.5 +/- 0.1 nmol/L per 10(6) cells/h for EP, PsA, and donors, respectively), increased copper/zinc-superoxide dismutase and catalase activities in granulocytes in EP patients and decreased in PsA patients, decreased activity of copper/zinc-superoxide dismutase (0.3 +/- 0.0, 1.8 +/- 0.1, and 2.2 +/- 0.2 U/mg protein for EP, PsA, and donors, respectively), and altered activity of catalase in psoriatic epidermis. Plasma levels of nitrites/nitrates were greater than normal in psoriatic patients. Supplementation resulted in significant improvement of clinical conditions, which corresponded to the faster versus placebo normalization of the oxidative stress markers. CONCLUSION: Supplementation with antioxidants coenzyme Q(10), vitamin E, and selenium could be feasible for the management of patients with severe forms of psoriasis.

Spring brings breezes, wheezes, and pollen oxidases.

While the release of pollen into the air is essential for the reproduction of plants, the accidental yet inevitable uptake of pollen into human airways can cause symptoms of seasonal allergies and asthma. The symptomatic response to pollen is caused by granulocytes that produce inflammation, which is due in part to oxidative stress through the action of NADPH oxidases. The recruitment of these inflammatory granulocytes was previously thought to depend entirely on the activation of an adaptive immune response. In this issue of the JCI, Boldogh et al. demonstrate that pollens contain endogenous NADPH oxidase activity, which functions to generate local "danger signals" in nearby airway epithelium. These signals in turn trigger the early recruitment of granulocytes, even in the absence of the adaptive immune response. These findings suggest that inhibition of the pollen oxidase may provide a way to antagonize allergic inflammation at a very early step.

Identification and characterization of CKLiK, a novel granulocyte Ca(++)/calmodulin-dependent kinase.

Human granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction-based strategy was used to identify novel granulocyte-specific kinases. A novel protein kinase complementary DNA with an open reading frame of 357 amino acids was identified with homology to calcium-calmodulin-dependent kinase I (CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of CKLiK messenger RNA (mRNA) expression in hematopoietic cells demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during neutrophilic differentiation of CD34(+) stem cells. CKLiK kinase activity was dependent on Ca(++) and calmodulin as analyzed by in vitro phosphorylation of cyclic adenosine monophosphate responsive element modulator (CREM). Furthermore, CKLiK- transfected cells treated with ionomycin demonstrated an induction of CRE- binding protein (CREB) transcriptional activity compared to control cells. Additionally, CaMK-kinasealpha enhanced CKLiK activity. In vivo activation of CKLiK was shown by addition of interleukin (IL)-8 to a myeloid cell line stably expressing CKLiK. Furthermore inducible activation of CKLiK was sufficient to induce extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase activity. These data identify a novel Ca(++)/calmodulin-dependent PMN- specific kinase that may play a role in Ca(++)-mediated regulation of human granulocyte functions.

Effects of organic and inorganic selenium supplementation on selenoenzyme activity in blood lymphocytes, granulocytes, platelets and erythrocytes.

The blood selenium (Se) concentration in the U.K. population has declined by approx. 50% between 1974 and 1991, reflecting a large decrease in dietary Se supply, with intakes only half the reference nutrient intake of 1 microg/kg body weight. Tissue levels of Se are readily influenced by dietary intake. Therefore selenoprotein activity may be sub-optimal due to low Se status, and thus compromise normal cell function. To examine the effects of changing Se intake on selenoproteins, we have determined the relative effectiveness of organic selenomethionine and inorganic sodium selenite (50 microg of Se daily for 28 days) in modulating glutathione peroxidase activities in blood cells from 45 healthy men and women, from a U.K. population. Transient and acute changes in lymphocyte, granulocyte and platelet phospholipid-hydroperoxide glutathione peroxidase (GPx4) activity occurred by day 7 or 14 of sodium selenite treatment and by day 7 in lymphocytes from selenomethionine-treated subjects compared with controls taking a placebo. In contrast, GPx4 activity in granulocytes and platelets in the selenomethionine group increased gradually over the 28 days. Cytosolic glutathione peroxidase (GPx1) activity in these blood cells from both treatment groups increased gradually over the 28 days. For each cellular selenoenzyme activity a significant inter-individual difference (P<0.001) in the extent of the response to Se supplementation was observed, but this was not related to blood Se concentrations either before or after treatments. Significant inverse correlations were evident between baseline enzyme activities and percentage change in activity after 28 days of supplementation [e.g. lymphocyte GPx4, r=-0.695 (P<0.001)], indicating that pre-treatment activity may be sub-optimal as a result of poor Se status. The different and contrasting effects that Se supplementation had on blood selenoenzyme activities may be indicative of a difference in metabolic need for Se regulated at the level of Se-dependent cell function.

Physical exercise-induced expression of inducible nitric oxide synthase and heme oxygenase-1 in human leukocytes: effects of RRR-alpha-tocopherol supplementation.

This study evaluated the effects of RRR-alpha-tocopherol (500 IU/day, 8 days) on in vivo cytokine response and cytoplasmic expression of inducible nitric oxide synthase (iNOS) and the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes after exhaustive exercise. Thirteen men were investigated in a double-blind, placebo-controlled, cross-over study with a wash-out period of 28 days. The exercise procedure consisted of an incremental treadmill test followed by a continuous run until exhaustion at 110% of the individual anaerobic threshold (total duration 28.5 +/- 0.8 min). HO-1 and iNOS protein were assessed in mono- (M), lympho-, and granulocytes (G) using flow cytometry. Plasma interleukin-6 (IL-6) and IL-8 were measured by ELISA. IL-6 rose significantly whereas IL-8 did not exhibit significant changes after exercise. Changes of IL-6 were not affected by RRR-alpha-tocopherol. Exercise induced an increase of iNOS protein primarily in M and G. A small, but significant, increase of HO-1 protein was measured in M and G. RRR-alpha-Tocopherol did not show any significant effects on cytoplasmic expression of iNOS and HO-1 at rest and after exercise. In conclusion, exhaustive exercise induces expression of iNOS and HO-1 in human leukocytes by a mechanism that is not sensitive to RRR-alpha-tocopherol supplementation.

Ginsenoside Rh2 and Rh3 induce differentiation of HL-60 cells into granulocytes: modulation of protein kinase C isoforms during differentiation by ginsenoside Rh2.

Ginsenoside Rh1 or Rh2 differentiated B16 melanoma or F9 teratocarnoma to phenotypic normal melanocyte-like cells or parietal endoderm-like cells. Ginsenoside Rh3 and Rh4 were recently isolated from Panax ginseng, but their biochemical and pharmacological effects remain unidentified. The present study investigated whether the ginsenoside Rh group (G-Rh1, -Rh2, -Rh3 and -Rh4) having similar structures induce differentiation of HL-60 cells and whether protein kinase C (PKC) is involved in differentiation by ginsenoside. Differentiation was assessed by Wright-Giemsa stain and nitroblue tetrazolium reduction. G-Rh2 and G-Rh3 induced differentiation of HL-60 cells into morphologically and functionally granulocytes but G-Rh1 and G-Rh4 did not. G-Rh2 and G-Rh3 arrested the cell cycle at the G1/S phase, consistent with the ability to induce differentiation in a decreasing order of retinoic acid > G-Rh2 > G-Rh3. During differentiation by G-Rh2, Ca2+/phospholipid-dependent PKC activity was increased in both the cytosol and total cell extract and Ca2+/phospholipid-dependent phosphorylation of 38 and 200 kDa endogenous proteins increased, while phosphorylation of 60, 64, 66 and 97 kDa proteins was Ca2+/phospholipid-independent. When cytosolic PKC isoforms were analyzed by immunoblotting, no significant change was observed in the alpha level, however, the immunoreactive 60 kDa band of a similar mass to the PKC catalytic fragment appeared following treatment with G-Rh2. The beta isoform was gradually increased with prolonged treatment. The gamma isoform was not detected in the cytosol of untreated cells, whereas a small amount was detected 5 days after treatment. It is concluded that G-Rh2 and G-Rh3 can induce differentiation of HL-60 cells into granulocytes and modulation of PKC isoform levels may contribute to differentiation of HL-60 cells by G-Rh2.

Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes.

Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12-LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

[Granulocyte alkaline phosphatase--a biomarker of chronic benzene exposure]. / Granulotsitna alkalna fosfataza--biomarker za khronichno benzolno vuzdeistvie.

In tracing the cellular population status in the peripheral blood of workers, exposed to benzene, was included and cytochemical determination of the alkaline phosphatase activity in leucocytes. This enzyme is accepted as marker of the neutrophilic granulocytes, as maturation of the cells and their antibacterial activity are parallel to the cytochemical activity of the enzyme. 78 workers from the coke-chemical production from state firm "Kremikovtsi" and 41 workers from the production "Benzene" and "Isopropylbenzene"--Oil Chemical Plant, Burgas are included. The benzene concentrations in the air of the working places in all productions are in the range of 5 to 50 mg/m3. For cytochemical determination of the alkaline phosphatase activity is used the method of L. Kaplow and phosphatase index was calculated. It was established that in 98.4% of all examined the alkaline phosphatase activity is inhibited to different rate, as from 46.5% [61 workers] it is zero. In considerably lower percentage of workers were established and other deviations: leucocytosis or leucopenia, neutropenia, increased percent of band neutrophils and toxic granules. The results of the investigation of the granulocyte population show that from all indices, the activity of granulocyte alkaline phosphatase demonstrates most convincing the early myelotoxic effect of benzene.

Cu/Zn superoxide dismutase activity does not parallel copper levels in copper supplemented HL-60 cells.

The objective of this research was to develop a method for measuring Cu/Zn-superoxide dismutase (Cu/Zn-SOD) (E.C. 1.15.1.1) in HL-60 cells and subsequently examine the relationship between cellular copper levels and the activity of this copper-requiring enzyme. In cells such as the neutrophil or HL-60 promyelocyte cell line, the activity of Cu/Zn-SOD cannot be measured because of an increase in the oxidation rate of the substrate by some unknown compound in the cells. Others have utilized heat treatment to inactivate the responsible compounds, however, we found that heat treatment of HL-60 cells resulted in a loss of over half of the activity of the enzyme. The method described here utilizes sodium azide to inhibit the substance(s) that are responsible for the enhanced rate of pyrogallol's oxidation. Gel filtration data confirmed that the compound responsible for the enhanced rate of pyrogallol oxidation was sensitive to azide and did not affect Cu/Zn-SOD activity. When HL-60 cells were incubated with various levels of copper, Cu/Zn-SOD activity did not reflect the cellular copper levels.

Ecto 5' nucleotidase (5'NT) as a sensitive indicator of human zinc deficiency.

Ecto 5' nucleotidase (5'NT) is an integral plasma membrane enzyme located on most mammalian cells, and it is zinc dependent. We assayed 5'NT activity in the lymphocytes of two groups of subjects. The first group of six subjects had a mild state of zinc deficiency, as measured on the basis of zinc levels in lymphocytes, granulocytes, and platelets, but were otherwise healthy. They received 50 mg zinc as acetate orally for 12 weeks. The second six subjects were normal human volunteers in whom a mild state of zinc deficiency was induced experimentally by dietary techniques (4.2 to 5.6 mg daily zinc intake). For the assay of 5'NT, intact lymphocytes were incubated with 8-14C-labeled inosine monophosphate as substrate. Product and substrate were separated by thin-layer chromatography. Zinc level in cells was measured by flameless atomic absorption technique. In the first group of subjects with zinc deficiency, the decreased activity of 5'NT was corrected and the cellular zinc levels were normalized by oral zinc supplementation (p less than 0.01). In the second group of subjects, the baseline data were compared with those in early zinc depletion (4 to 8 weeks) and late depletion periods (greater than 20 weeks). A decrease in the activity of 5'NT was observed during the early zinc depletion phase. Zinc levels in lymphocytes, granulocytes, and platelets decreased significantly only during the late zinc depletion phase (p less than 0.01). Plasma zinc level did not change even during the late zinc depletion phase. Our studies show that 5'NT activity may be a sensitive and useful indicator of mild deficiency of zinc in human subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione peroxidase and selenium deficiency in patients receiving home parenteral nutrition: time course for development of deficiency and repletion of enzyme activity in plasma and blood cells.

The time course for the depletion of red blood cell (RBC) and plasma glutathione peroxidase (GSHPx) activity in five patients receiving home parenteral nutrition was documented. During the development of the RBC GSHPx deficiency, enzymatic activity and protein content decreased. Plasma and RBC selenium content were similarly decreased as was cellular metabolism of exogenous hydrogen peroxide. When the replacement of Se (selenious acid) began, there was a rapid increase (within 6 h) in plasma GSHPx activity. Platelet and granulocyte (PMN) GSHPx activity, which were low when Se replacement began, became normal within a time consistent with the kinetics of platelet and PMN production. RBC GSHPx did not become normal for 3-4 mo. This is consistent with the time course for RBC production. It appears that the repletion of blood cell GSHPx requires the formation of these cells in the presence of Se.

Glutathione peroxidase activity in patients with rheumatoid arthritis and in normal subjects: effects of long-term selenium supplementation.

The effects of dietary supplementation with selenium were studied in 6 patients with severe, active rheumatoid arthritis (RA) and in 6 healthy control subjects. Initial concentrations of Se in red blood cells and in serum, and the activity of the Se-dependent enzyme glutathione peroxidase (GSH-Px) in red blood cells, serum, and granulocytes were significantly lower in RA patients compared with controls. During Se supplementation, however, the differences in Se levels and in GSH-Px activity between the 2 groups disappeared, except that, in RA patients, GSH-Px activity in granulocytes increased but remained significantly lower than in controls.