Human Blood Leukocytes Research Articles

The regulation of CD11b integrin levels on human blood leukocytes and leukotriene B4-stimulated skin by a specific leukotriene B4 receptor antagonist (LY293111)

CD11b is part of the β2-integrin Mac-1 and plays an important role in neutrophil adhesion. Leukotriene B 4 (LTB 4) is an active upregulator of neutrophil CD11b-expression, acts as a potent chemoattractant to neutrophils and is also known to upmodulate epidermal proliferation. We performed a placebo-controlled study on LY293111, an oral LTB 4 receptor antagonist. Twenty healthy male volunteers were randomised over three treatment groups that received placebo, 48 mg, or 200 mg drug twice daily for 10 days. Before and after treatment, flow cytometrical CD11b assessment was performed on in vitro LTB 4-stimulated peripheral blood neutrophils. Additionally, skin biopsies were taken at 24 and 72 h after epicutaneous LTB 4 application, before and after treatment. The effects on skin were assessed immunohistochemically using various markers. All observed effects were dose related. CD11b upregulation on blood neutrophils was significantly suppressed in both treatment groups compared to placebo. In skin, a significant suppression of inflammation and hyperproliferation occurred. Pronounced inhibition was observed on neutrophil migration into the epidermis and the inflammatory infiltrate was decreased. A similar but weaker response was seen in the dermis. The number of cycling cells as well as suprabasal keratin-16 expression were decreased in both treatment groups. LY293111 proved to be a potent inhibitor of LTB 4-induced cutaneous inflammation and hyperproliferation. The potent antiinflammatory effect in vivo and the fact that in the present study the compound showed no clinically significant side effects make it an interesting drug in the future treatment of inflammatory conditions predominated by neutrophils.

Human NK cell-mediated direct and IgG-dependent cytotoxicity against xenogeneic porcine endothelial cells

Once hyperacute rejection has been prevented, the pig-to-human xenograft might be exposed to vascular cell-mediated rejection directed against vascular structures. In order to evaluate the relative importance of direct and antibody-dependent anti-endothelial cell-mediated cytotoxicity in different individuals, freshly isolated human blood leukocytes were incubated with confluent porcine aortic endothelial cells (PAEC) in a 4 h Cr-release cytotoxicity assay. Peripheral blood mononuclear cells (PBMC) and lymphocytes (PBL) of all subjects tested (but not monocytes or neutrophils) directly killed PAEC, with wide interindividual variations (from 2.8% to 32%). The addition of heat-inactivated autologous serum to PBMC and PBL (but not to myeloid cells) always enhanced cytotoxicity. This antibody-dependent cell-mediated cytotoxicity (ADCC) was also observed in the presence of adult pooled serum and cord blood pooled serum and was eliminated after adsorption of adult pooled serum to insoluble protein A, demonstrating that IgG is the only class of immunoglobulin involved in this phenomenon. Moreover, blocking FcγRIII with an anti-CD16 mAb eliminated ADCC without affecting direct cytotoxicity. When the ADCC exerted by the PBL of all subjects was assessed with the same preparation of purified IgG, wide interindividual variations were again observed. Surprisingly, there was no correlation between direct cytotoxicity and ADCC although, as depletion experiments demonstrated, both were due to CD16 + natural killer (NK) cells. These results argue that CD16 + NK cells could play an important role in early vascular rejection of porcine discordant xenografts, by both a direct and an IgG xenoreactive natural antibody-dependent cell-mediated cytotoxicity.

Flow cytometry and staining kinetics to monitor the G0/G1 transition

The phenanthridine dyes propidium iodide (PI) and ethidium bromide (EB) have contributed to make DNA quantitation a routine measurement by flow cytometry. As planar molecules they easily intercalate between base pairs of double stranded nucleic acids. When the quantity of dye exceeds the amount of DNA, the intensity of emitted fluorescence can be related to the amount of DNA. This is, in fact, the basic requirement of quantitative measurement. When staining is performed at low dye concentration and there are no sufficient molecules to saturate all the available DNA-binding sites, the emitted fluorescence, other than the true DNA quantity, may be dependent on the state of chromatin condensation. A staining procedure aimed to stress the influence of nuclear structure on the emitted fluorescence of PI is described. This is achieved by means of a very low dye concentration (<0.01 μg/ml PI) to guarantee staining far below saturation condition. In this particular condition the staining rate slows down to be monitored by flow cytometry. As different experimental models the leucocytes of the normal human peripheral blood have been used taking into account the relatively condensed chromatin of granulocytes with respect to that of the mononuclear cells. Significantly higher fluorescence intensity have been obtained from mononuclear cells compared to that of the polymorphonuclear ones. Quiescent versus exponentially proliferating cell cultures had also been tested. In this staining condition low fluorescence intensity have been obtained by condensed chromatin structure (G0) in comparison to the more decondensed (G1) one.

Cloning, Functional Expression and Tissue Distribution of the Human P2Y6Receptor

In order to isolate new subtypes of P2Y purinoceptors, a human placenta cDNA library was screened at middle stringency with a P2Y4probe. The purification and the sequencing of several clones led us to identify a 984 base pair open reading frame encoding a new human P2Y receptor. It appeared later that this sequence corresponds to the human ortholog (88% amino acid identity) of the rat receptor recently cloned by Chang et al (J. Biol. Chem.270,26152–26158, 1995) and called P2Y6. Northern blot analysis detected human P2Y6receptor messenger RNA in human spleen, placenta, thymus, intestine, and blood leukocytes. In 1321N1 cells stably expressing the human P2Y6receptor, the formation of IP3was stimulated by nucleotides with the following order of potency: UDP>5-bromo-UPT>UTP>ADP>2-methylthio-ATP>ATP. The P2Y6receptor, together with the previously cloned P2Y4subtype (Communi et al.,J. Biol. Chem.,270,30849–30852, 1995), belongs thus to a subfamily of pyrimidinoceptors inside the P2Y family.

5-Lipoxygenase inhibition by acetyl-11-keto-β-boswellic acid (AKBA) by a novel mechanism

Acetyl-11-keto-β-boswellic acid (AKAB) from Boswellia serrata and B. carterii acts directly on purified 5-lipoxygenase of human blood leukocytes at a selective site for pentacyclic triterpenes that is different from the arachidonate substrate binding site. The pentacyclic triterpene ring is crucial for binding to the enzyme, whereas functional groups (11-keto function in addition to a hydrophilic group on C 4 of ring A) are essential for the 5-lipoxygenase activity.

The effect of recombinant human colony stimulating factor (G-CSF) on the function of human blood and urinary leukocytes

To study the function of leukocyte in urinary tract infection (UTI), the effect of granulocyte colony-stimulating factor (G-CSF) and various osmolarities on the function were studied. The superoxide production of blood leukocytes increased by treatment of G-CSF with its dose dependency. This effect was not observed in urinary leukocytes. This increased function was well preserved even in higher osmotic atmosphere when previously treated by the G-CSF. Therefore, urinary leukocyte activated by G-CSF and migrating from the blood through the tissue may still have its function and work in the patients with UTI.

Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity.

Group B streptococci (GBS) cause sepsis and meningitis in neonates and serious infections in adults with underlying chronic illnesses. Specific antibodies have been shown to be an important factor in protective immunity for neonates, but the role of serum complement is less well defined. To elucidate the function of the complement system in immunity to this pathogen, we have used the approach of gene targeting in embryonic stem cells to generate mice totally deficient in complement component C3. Comparison of C3-deficient mice with mice deficient in complement component C4 demonstrated that the 50% lethal dose for GBS infection was reduced by approximately 50-fold and 25-fold, respectively, compared to control mice. GBS were effectively killed in vitro by human blood leukocytes in the presence of specific antibody and C4-deficient serum but not C3-deficient serum. The defective opsonization by C3-deficient serum in vitro was corroborated by in vivo studies in which passive immunization of pregnant dams with specific antibodies conferred protection from GBS challenge to normal and C4-deficient pups but not C3-deficient pups. These results indicate that the alternative pathway is sufficient to mediate effective opsonophagocytosis and protective immunity to GBS in the presence of specific antibody. In contrast, the increased susceptibility to infection of non-immune mice deficient in either C3 or C4 implies that the classical pathway plays an essential role in host defense against GBS infection in the absence of specific immunity.

Interactions of pulmonary surfactant protein D (SP-D) with human blood leukocytes.

Surfactant protein D (SP-D) is believed to contribute to nonimmune host defense within the alveoli and distal airways of the lung. SP-D molecules can bind to specific carbohydrates on the surface of bacterial, fungal, and viral organisms and can also interact with membrane glycoconjugates expressed by alveolar macrophages. Because neutrophils (PMN) and monocytes are recruited into the airspaces in association with many types of infection or lung injury, we examined the interactions of these cells with purified natural and recombinant SP-Ds, using a modified Boyden chamber assay and checkerboard analysis. Natural or recombinant rat SP-D (approximately 10(-9) to 10(-13) M) showed dose-dependent effects on human PMN and monocyte migration with a maximal response at a SP-D concentration of 5 ng/ml (approximately 10(-11) M). The migratory response was comparable to that obtained with the optimum concentration of FMLP (10(-8) M). HL-60 cells, after induction of differentiation with DMSO, responded to SP-D with the same dose-response as neutrophils. The effects of SP-D were abrogated by the simultaneous addition of SP-D to the upper chamber or by the addition of antibodies to the carboxy-terminal lectin domain. Migration toward SP-D was markedly inhibited (< 10% of controls) by 10 mM maltose but was not significantly inhibited by to 50 mM lactose. These studies establish that SP-D can bind to specific sites on neutrophils and monocytes and strongly suggest that these interactions involve the saccharide binding domains of SP-D.

Study of antioxidant properties of metal aspartates.

The effects of Zn, Mg, Cr, Cu, and Mn aspartates, their commercial formulation Inzolen, and the individual commercial medicine Unizinc, on oxygen radical production by enzymes [xanthine oxidase, horseradish peroxidase, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase] and phagocytic cells (human blood leukocytes) have been studied. The formation of oxygen radicals was measured by luminol- and lucigenin-amplified chemiluminescence and by the reduction of cytochrome c. All these compounds (excluding Cr aspartate) turn out to be inhibitors of oxygen radical formation in the systems studied (excluding horseradish peroxidase). Their inhibitory activities were a consequence of both the scavenging of free radicals and the inhibition of xanthine oxidase and NADPH oxidase activities. As expected, the most active free-radical scavengers were transition metal Cu and Mn aspartates, which mimicked the activities of copper-zinc and manganese dismutases. However, surprisingly non-transition metal Zn and Mg aspartates were also able to scavenge oxygen radicals. It was suggested that the scavenging activities of Zn and Mg aspartates may be explained by affecting the rate of spontaneous dismutation of the superoxide ion. In addition, it was found that Zn aspartate is an efficient inhibitor of the formation of the most reactive hydroxyl radicals. These antioxidant properties of Zn aspartate make it important in medicine for the prevention and treatment of free radical pathologies.

Absorption and blood leukocyte dynamics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) from intranasally administered preparations containing rhG-CSF and cyclodextrins in rabbits

The absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and blood leukocyte dynamics following intranasal (i.n.) administration of aqueous preparations containing rhG-CSF with or without cyclodextrins (CyDs) were investigated in conscious rabbits by measuring the serum G-CSF concentration and the total number of leukocytes in peripheral blood. Serum G-CSF concentration was rapidly increased by coadministration of rhG-CSF and CyD, particularly, α and β types. The total number of leukocytes in blood peripheral blood also increased (the maximum of level of approx./μl was attained 24 h after administration) and exceeded physiological levels (5200–12000/μl) in rabbits. The increase in the number of leukocytes was observed between approx. 8 and 48 h after i.n. administration. These results suggest that rhG-CSF is efficiently absorbed through the nasal mucosa. It may be concluded that the development of a rhG-CSF delivery system could be achieved in the form of the nasal administration of rhG-CSF with CyDs.

Human chorionic gonadotrophin: a chemoattractant for human blood monocytes, neutrophils and lymphocytes

Treatment of rats with human chorionic gonadotrophin (hCG) induced in the testes an inflammation-like reaction characterized by migration of leukocytes into the interstitial space. In order to find out whether hCG acts in a direct manner in this process, we tested peripheral human blood leukocyte attraction by hCG in vitro. Chemotaxis through cellulose nitrate to gradients of test substances was measured using a 48-well microchemotaxis chamber. Human CG was found to be a potent attractor of neutrophils, monocytes and lymphocytes in vitro in the picomolar concentration range. Checkerboard analyses revealed that the type of migration depends on positive concentration gradients of hCG. The chemoattractant nature of hCG is consistent with its having a role to play in regulation of tissue accumulation of these cells within the reproductive tract.

Human immunodeficiency virus type 1 infection of neonatal severe combined immunodeficient mice xenografted with human cord blood cells.

In these studies, neonatal C.B-17 severe combined immunodeficient (nSCID) mice were reconstituted with human cord blood leukocytes (hu-CBLs). The resulting hu-CBL-nSCID mice contained readily detectable human CD3+ T lymphocytes and CD20+ human B cells, and produced substantial levels of human IgM and IgG (including all subclasses). Human cells persisted in lymphoid organs and peripheral blood for at least 8 weeks, and CD4+ T cells outnumbered CD8+ T cells. Engraftment of human cells in peripheral lymphoid organs and blood was much greater than that seen in adult SCID mice grafted with adult peripheral blood leukocytes (PBLs). Hu-CBL-nSCID mice were susceptible to infection with laboratory-adapted and fresh clinical human immunodeficiency virus type 1 (HIV-1) isolates. Following infection with HIV-1, virus could be recovered by the coculture of spleen, lymph node, peritoneal cavity, liver, and plasma samples from hu-CBL-nSCID mice with fresh human peripheral blood mononuclear cells, and proviral copies were detectable following amplification using the polymerase chain reaction (PCR). HIV p24 core antigen levels in hu-CBL-nSCID mouse plasma were consistent with ongoing viral replication and high viral burdens. Rapid CD4+ T cell depletion occurred following infection with laboratory isolates of HIV-1 or a syncytium-inducing clinical isolate, but a non-syncytium-inducing clinical isolate caused expansion of CD8+ T cells, leading to an inversion of the CD4:CD8 ratio with only a transient decrease in CD4+ T cells. These results suggest that the hu-CBL-nSCID mouse system has unique features that mimic certain aspects of pediatric HIV infection, and distinguish it from other animal models of HIV infection, including the related hu-PBL-SCID model.

Interleukin-1 Regulates the Proliferation of Leukocytes in Human Corneal Cell-Peripheral Blood Leukocyte Cocultures

We have documented the inability of human corneal epithelial-like cells to suppress proliferation of peripheral blood leukocytes (PBLs) induced by allogeneic PBLs in a mixed leukocyte reaction (MLR). Instead, enhanced proliferation of PBLs, albeit small, was consistently noted as indicated by uptake of radiolabeled thymidine. Maximum proliferation of PBLs was detected when a mixed leukocyte reaction (MLR) was conducted in the presence of corneal cells. High levels of interleukin-1 beta (IL-1 beta) were found during MLR irrespective of the presence of corneal cells. High levels of IL-1 beta correlated well with observed synergistic stimulation of PBL proliferation by corneal cells and stimulating allogeneic PBLs. In PBL-corneal cell cocultures, PBLs produced IL-1 beta; corneal cells contributed large amounts of prostaglandin E2 (PGE2). Although indomethacin completely blocked prostaglandin E2 production, it did not significantly alter the results. Our data show that PBLs and corneal cells can reciprocate each other's presence, and, under appropriate conditions, corneal cells can deliver at least one signal to enhance rather than suppress antigen-driven PBL proliferation. Our data suggest a role for immunoregulatory cytokines and prostanoids such as IL-1 beta and PGE2 in these interactions.

Comparative Study of the Effects of Cefodizime and HBW 538 in Potentiating the Production of Reactive Oxygen Species by Human Polymorphonuclear Leukocytes

The immunomodulatory activity of cefodizime (CDZM), an aminothiazolylcephalosporin, was compared to that of HBW 538, a derivative of the CDZM side chain at position 3 (the mercaptothiazolyl group) in respect to the production of reactive oxygen species (ROS) by human whole blood and polymorphonuclear leukocytes (PMN) in vitro. Ten-fold diluted whole blood and PMN from healthy individuals were incubated with CDZM or HBW 538 alone at the concentrations of 1, 10, or 100 micrograms/ml, or CDZM or HBW 538 at 100 micrograms/ml in combination with tumor necrosis factor-alpha (TNF-alpha) at 100 U/ml or lipopolysaccharide (LPS) at 1 microgram/ml. The production of ROS was measured by a chemiluminescence (CL) assay in which luminol was added to a mixture and after which the PMN or whole blood were stimulated with nonopsonized zymosan or phorbol myristate acetate. The following results were obtained: (1) The CL responses of whole blood and PMN were slightly but not significantly enhanced by CDZM at 100 micrograms/ml, whereas both CL responses were significantly enhanced by exposure to HBW 538 at 10 and 100 micrograms/ml. (2) The enhanced PMN CL response which followed priming with TNF-alpha or LPS was not augmented by CDZM but was significantly augmented by HBW 538. These results indicate that the ability of the HBW 538 molecule to enhance the production of ROS by stimulated PMN and to act agonistically with TNF-alpha or LPS is abrogated when HBW 538 is part of the CDZM molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of endopeptidase 24.11 and endopeptidase 24.15 in human blood leukocytes.

Endopeptidase 24.11 (EP 24.11; also called neutral endopeptidase, enkephalinase, CALLA, or CD10) and endopeptidase 24.15 (EP 24.15) are widely distributed neutral metalloendopeptidases that degrade a number of bioactive peptides including substance P, bradykinin, neurotensin, and chemotactic peptides. In this study we used sensitive substrates and specific inhibitors to quantitate the levels of these enzymes in purified peripheral human blood leukocytes obtained from healthy blood donors. We found that neutrophils did not contain detectable amounts of EP 24.15. In contrast, T lymphocytes, B lymphocytes, and monocytes contained significant amounts of the enzyme (446 +/- 248,314 +/- 183, and 484 +/- 212 nmol/mg protein/h, respectively). Neutrophils contained significant amounts of EP 24.11 (266 +/- 130 nmol/mg protein/h). Significantly lower levels of the enzyme were found in T lymphocytes, B lymphocytes, and monocytes (94 +/- 31, 87 +/- 38, and 20 +/- 13 nmol/mg protein/h, respectively). These findings suggest that the effects of some bioactive peptides on peripheral blood leukocyte function may be modulated by these enzymes.

Stimulation of protective antibodies against type Ia and Ib group B streptococci by a type Ia polysaccharide-tetanus toxoid conjugate vaccine.

Antisera elicited by type Ia group B streptococci (GBS) contain antibodies that react with both type Ia and type Ib strains. Previous studies suggested that antibodies elicited by type Ia organisms recognized a carbohydrate antigen or epitope common to Ia and Ib strains. We now report the synthesis and immunogenicity testing of a type Ia polysaccharide-tetanus toxoid (Ia-TT) conjugate vaccine. Ia-TT elicited type Ia polysaccharide-specific immunoglobulin G antibodies in all three of the rabbits inoculated. In competitive enzyme-linked immunosorbent assay, these antibodies reacted with high affinity to type Ia polysaccharide and with lower affinity to the structurally related GBS type Ib polysaccharide. Despite the lower binding affinity of the Ia-TT-induced antibodies for the type Ib polysaccharide, Ia-TT antiserum opsonized not only type Ia GBS but also type Ib GBS for killing by human blood leukocytes. Ia-TT antiserum was also evaluated in a mouse model designed to test the efficacy of maternal antibodies in protecting neonates against GBS infection. Pups born to dams that had received Ia-TT antiserum were protected against lethal challenge with either type Ia or Ib GBS. These studies using a polysaccharide-protein conjugate as an immunogen support the view that the carbohydrate immunodeterminant recognized on Ib strains by Ia antisera is a common epitope contained within the structurally related Ia and Ib capsular polysaccharides. Although antibodies elicited by Ia-TT had protective activity against both Ia and Ib strains, these antibodies reacted with lower affinity to Ib than to Ia polysaccharide.

An analysis of γ-ray-induced DNA damage in human blood leukocytes, lymphocytes and granulocytes

Lymphocytes and granulocytes were separated from human peripheral blood and irradiated with low doses of γ-rays (0.05–0.5 Gy) from a 137Cs source. The magnitude and intercellular distribution of DNA damage, i.e., single-strand breaks and alkali-labile lesions, were compared with those obtained in unfractionated leukocytes irradiated in whole blood, using the alkaline single-cell gel-electrophoresis technique. Based on the extent of DNA migration, irradiation resulted in a linear and dose-dependent increase in DNA damage in all 3 cell populations, with a significant increase being detected at 0.05 Gy. The dose-dependent increase for DNA migration was not significantly different between separated lymphocytes and granulocytes, but their responses were significantly elevated over that obtained for leukocytes irradiated in whole blood. Based on an analysis of the ration of the range to the standard deviation for each cell population at each dose of radiation, the distribution of damage among cells was relatively homogeneous and independent of dose and cell population. These results are consistent with a hypothesis that irradiation of leukocytes in whole blood partially protects against radical-induced DNA damage.

Expression of HERV-K Proviruses in Human Leukocytes

HERV-K is a 50-copy, human endogenous, class 1 retroviral element that contains some polycistrons with gag, pol, and env open reading frames. Although expression of HERV-K proviruses has been shown in cultured human cell lines, expression of these elements has not been shown in human blood leukocytes. Using both reverse transcriptase-polymerase chain reaction and ribonuclease protection techniques, we show HERV-K pol gene expression in human blood leukocytes. Expression in blood leukocytes from 7 normal individuals was from a variety of different HERV-K proviruses, while restricted expression was observed in blood cells of 5 leukemia patients and 3 polycythemia vera patients. Evidence is presented suggesting that the restricted expression in leukemia blood cells is a result of gene regulation, not gene amplification.

Expression of HERV-K proviruses in human leukocytes

HERV-K is a 50-copy, human endogenous, class 1 retroviral element that contains some polycistrons with gag, pol, and env open reading frames. Although expression of HERV-K proviruses has been shown in cultured human cell lines, expression of these elements has not been shown in human blood leukocytes. Using both reverse transcriptase-polymerase chain reaction and ribonuclease protection techniques, we show HERV-K pol gene expression in human blood leukocytes. Expression in blood leukocytes from 7 normal individuals was from a variety of different HERV-K proviruses, while restricted expression was observed in blood cells of 5 leukemia patients and 3 polycythemia vera patients. Evidence is presented suggesting that the restricted expression in leukemia blood cells is a result of gene regulation, not gene amplification.

Expression of human endogenous retrovirus (HERV-K) in chronic myeloid leukemia.

We have previously demonstrated the presence of a reverse transcriptase-like enzyme in retroviral particles from patients with essential thrombocythemia, polycythemia vera, and chronic myelogenous leukemia. It was subsequently shown that the human genome contains 50 copies of HERV-K. HERV-K is a human endogenous class I retroviral element that contains gag, pol, and env open reading frames. Using both reverse transcriptase-polymerase chain reaction and ribonuclease protection assays, it is demonstrated that the HERV-K pol is expressed in human blood leukocytes. The data indicates that this expression is restricted in CML white cells and is the result of gene regulation.