Response Of Human Polymorphonuclear Leukocytes Research Articles

Effects of interleukin-15 on antifungal responses of human polymorphonuclear leukocytes against Fusarium spp. and Scedosporium spp.

Fusarium spp. and Scedosporium spp. have emerged as important fungal pathogens that are frequently resistant to antifungal compounds. We investigated the effects of human interleukin-15 (IL-15) on human polymorphonuclear leukocyte (PMNL) activity against Fusarium solani and Fusarium oxysporum as well as Scedosporium prolificans and Scedosporium apiospermum. IL-15 (100 ng/ml) significantly enhanced PMNL-induced hyphal damage of both Fusarium spp. and S. prolificans after incubation for 22 h ( P < 0.01 ) but not S. apiospermum. In addition, IL-15 enhanced PMNL oxidative respiratory burst evaluated as superoxide anion production in response to S. prolificans but not to the other fungi after 2 h incubation. IL-15 increased interleukin-8 (IL-8) release from PMNLs challenged with hyphae of F. solani and S. prolificans ( P≤ 0.04 ). Release of tumor necrosis factor-α was not affected. The species-dependent enhancement of hyphal damage and induction of IL-8 release suggest that IL-15 plays an important role in the immunomodulation of host response to these emerging fungal pathogens.

Invasive infection with Trichosporon inkin in 2 siblings with chronic granulomatous disease

A 9-year-old girl with autosomal recessive chronic granulomatous disease (CGD) presented with asymptomatic bilateral pulmonary infiltrates on routine computed tomography. Fine-needle aspirate of the infiltrates was obtained and showed fungal cells resembling Trichosporon inkin . The specimen grew in culture, and testing by means of both API 20C and PCR amplification confirmed the diagnosis of T inkin . The infiltrates increased in size, despite sequential therapy with voriconazole, liposomal amphotericin B, caspofungin, and posaconazole. The patient required resection of the infected lung tissue, after which she recovered completely. While she was undergoing therapy, her 13-year-old brother, also with CGD, was given a diagnosis of bilateral T inkin -induced pulmonary infection. He also required bilateral pulmonary resection for cure. These cases demonstrate the predisposition of patients with CGD to have invasive infections with unusual fungal organisms, such as T inkin . They also illustrate the difficulty of treating invasive T inkin infections with antifungal agents alone. There are 9 previously reported cases of invasive infections caused by T inkin , 3 of which are in patients with CGD. All patients required removal of infected prosthetic devices or surgical resection of infected tissue for cure.

Immunoglobulin enhances the bioactive-glass-induced chemiluminescence response of human polymorphonuclear leukocytes.

Bioactive glasses are bone substitutes that chemically bind to bone. Implanted materials always elicit a response from surrounding tissues and thereby can activate inflammatory cells, with subsequent release of biomaterial and tissue-damaging agents. Bioactive glasses can activate polymorphonuclear leukocytes (PMNL) and induce a release of reactive oxygen metabolites (ROM). Adsorption of proteins on the surface of the implanted material may influence the subsequent inflammatory cell response. The effect of Sandoglobulin(R) (SG) and albumin on the ROM release by PMNLs induced by a bioactive glass was studied by a chemiluminescence (CL) assay. An enhanced effect for SG and inhibitory effect for albumin on the CL response of PMNLs was observable. The CL response of the PMNLs was dependent on the incubation time of the glass in solution.

Mechanisms of the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide-stimulated human neutrophils.

The goal of this study was to explain the priming effect of lipopolysaccharides (LPS) in human polymorphonuclear leukocytes on leukotriene B4 (LTB4) biosynthesis after stimulation with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This priming effect for LTB4 biosynthesis was maximal after a 30 min preincubation with LPS but was lost when incubations were extended to 90 min or longer. Priming with LPS resulted in an enhanced maximal activation of 5-lipoxygenase (5- to15-fold above unprimed cells) as well as a prolonged activation of the enzyme after stimulation with fMLP compared to that measured in unprimed cells. The activation of 5-lipoxygenase was associated with its translocation to the nuclear fraction of the cell after stimulation of LPS-primed cells but not of unprimed cells. Priming of cells with LPS also resulted in an enhanced capacity (fivefold increase) for arachidonic acid (AA) release after stimulation with fMLP compared to unprimed cells as measured by mass spectrometry. This release of AA was very efficiently blocked in a dose-dependent manner by the 85 kDa cytosolic phospholipase A2 (PLA2) inhibitor MAFP (IC50=10nM) but not by the 14 kDa secretory PLA2 inhibitor SB 203347 (up to 5 microM), indicating that the 85 kDa cPLA2 is the PLA2 responsible for AA release in response to receptor-mediated agonists. In accord with inhibitor studies, the LPS-mediated phosphorylation of cPLA2 followed the same kinetics as the priming for AA release, and a measurable fMLP-induced translocation of cPLA2 was observed only in primed cells. As with AA release and LTB4 biosynthesis, both the phosphorylation and capacity to translocate cPLA2 were reversed when the preincubation period with LPS was extended to 120 min. These results explain some of the cellular events responsible for the potentiation and subsequent decline of functional responses of human polymorphonuclear leukocytes recruited to inflammatory foci.

Azurophilic granules of human neutrophils contain CD14.

CD14, the leukocyte receptor for lipopolysaccharide (LPS), is important in the response of human polymorphonuclear leukocytes (PMNs) to infection with gram-negative bacteria. The level of CD14 on the PMN surface increases after exposure to some inflammatory stimuli such as N-formyl-methionyl-leucyl-phenylalanine (fMLP). These newly expressed CD14 molecules probably come from an intracellular pool of preformed receptors. We sought to further characterize PMN CD14 expression, upregulation, and shedding and to define the intracellular location of CD14 molecules. Our results demonstrate that both LPS and fMLP significantly increased CD14 cell surface expression; however, neither phorbol myristate acetate (PMA) or A23187 increased receptor levels on the PMN surface. Neither fMLP, PMA, or A23187 stimulated the release of soluble CD14 from PMNs. Intracellular CD14 was observed in >90% of PMNs examined by flow cytometry and confocal microscopy. Additional analyses using CD14 enzyme-linked immunosorbent assays and electron microscopy studies, examining PMN granules separated by discontinuous sucrose or Percoll gradients, showed that CD14 was present in both the plasma membrane-secretory vesicle fractions and azurophilic granules.

Suppression of Leukotriene B4 Biosynthesis by Endogenous Adenosine in Ligand-activated Human Neutrophils

Adenosine (Ado) has been shown to suppress several functional responses of human polymorphonuclear leukocytes (PMNs). The current study investigated whether endogenous Ado regulates the biosynthesis of leukotriene (LT)B4 in ligand-stimulated PMNs. Measurements of Ado in PMN resuspended in Hanks' buffered salt solution (HBSS) or plasma showed a cell concentration- and time-dependent accumulation of the nucleoside. The removal of endogenous Ado with either Ado deaminase or the blockade of its action by the Ado A2a receptor antagonist, 8-(3-chlorostyryl) caffeine, markedly increased LTB4 biosynthesis upon ligand stimulation in HBSS. Similarly, LTB4 synthesis by ligand-stimulated PMNs in plasma (containing recombinant LTA4 hydrolase to allow the conversion of protein-bound LTA4) was strongly enhanced by addition of Ado deaminase. Addition of red blood cells to suspensions of PMNs in plasma mimicked the effect of adding Ado deaminase and LTA4 hydrolase in enhancing LTB4 biosynthesis upon ligand stimulation. This effect of red blood cells on LTB4 biosynthesis was blocked by dipyridamole, an inhibitor of Ado transport, or captopril, an inhibitor of LTA4 hydrolase. These results demonstrate that endogenous Ado efficiently downregulates ligand-stimulated LTB4 biosynthesis in PMN suspensions, pointing out a potentially important regulatory function of Ado in inflammatory exudates. These results also unveil a dual role for red blood cells in upregulating LTB4 biosynthesis, namely, the removal of endogenous Ado and the conversion of LTA4 released by activated PMNs.

Characterization of the chemiluminescence response of polymorphonuclear leukocytes stimulated with urate crystals.

We studied the luminol-amplified chemiluminescence (CL) response of human polymorphonuclear leukocytes (PMNLs) stimulated with synthetic monosodium urate monohydrate (MSUM) crystals. Stimulation of PMNLs prepared from healthy controls with MSUM resulted in a bimodal CL response with two peaks after 1 min and 4-6 min, respectively. MSUM caused neutrophils to produce reactive oxygen species (ROS), which are released from the cells as well as ROS with an intracellular localization. Proportionally more of the CL activity generated during the first peak was of extracellular origin. The MSUM-induced light-generating mechanism was azide-sensitive, required superoxide anion and was strongly suppressed by horseradish peroxidase.

Characterization of the Chemiluminescence Response of Polymorphonuclear Leukocytes Stimulated with Urate Crystals

We studied the luminol-amplified chemiluminescence (CL) response of human polymorphonuclear leukocytes (PMNLs) stimulated with synthetic monosodium urate monohydrate (MSUM) crystals. Stimulation of PMNLs prepared from healthy controls with MSUM resulted in a bimodal CL response with two peaks after 1 min and 4–6 min, respectively. MSUM caused neutrophils to produce reactive oxygen species (ROS), which are released from the cells as well as ROS with an intracellular localization. Proportionally more of the CL activity generated during the first peak was of extracellular origin. The MSUM-induced light-generating mechanism was azide-sensitive, required superoxide anion and was strongly suppressed by horseradish peroxidase.

Pharmacological control of human polymorphonuclear leukocyte degranulation by fenamates and inhibitors of receptor-mediated calcium entry and protein kinase C

The present work was designed to study the mechanism of inhibitory action of flufenamic and tolfenamic acids on the degranulation response of human polymorphonuclear leukocytes (PMNs). We have recently shown that fenamates inhibit PMN degranulation as well as other PMN functions at micromolar drug concentrations. However, the mechanism of their action remains unknown. To clarify this mechanism, the degranulation response was induced by agents known to activate different steps in the activation cascade in PMNs: the receptor-mediated activator fMLP ( N-formyl-L-methionyl-L-leucyl-L-phenylalanine); a calcium ionophore (A23187); an inhibitor of calcium-ATPase (thapsigargin); and an activator of protein kinase C (phorbol myristate acetate, PMA). For comparison, SK&F 96365 (an inhibitor of receptor-mediated calcium entry), Ro 31-8220 (an inhibitor of protein kinase C) and ketoprofen (another cyclooxygenase inhibitor) were used. Flufenamic and tolfenamic acids inhibited A23187- and fMLP-induced degranulation in a dose-dependent manner. The thapsigargin-triggered response was reduced only slightly and that induced by PMA remained unaltered. The pattern of the inhibitory action of fenamates differed from those of Ro 31-8220 and ketoprofen. The action of fenamates resembled that of the inhibitor of receptor-mediated calcium entry, SK&F 96365, especially when A23187, fMLP or PMA were used to stimulate the cells. This prompted us to measure the effects of flufenamic and tolfenamic acids on receptor-mediated calcium entry. The two fenamates inhibited the fMLP-induced increase in intracellular free calcium in fura-2 loaded PMNs in the presence but not in the absence of extracellular calcium. The results suggest that the suppressive actions of fenamates on PMN degranulation are neither related to the activity of cyclooxygenase nor PMA-activated protein kinase C. In contrast, fenamates resemble the antagonist of receptor-mediated calcium entry, SK&F 96365, in their antagonistic action on PMN degranulation.

Hemolytically Inactive C5b67 Complex: An Agonist of Polymorphonuclear Leukocytes

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.

Role of YadA in resistance of Yersinia enterocolitica to phagocytosis by human polymorphonuclear leukocytes

Pathogenic Yersinia enterocolitica cells do not induce the chemiluminescence response of human polymorphonuclear leukocytes (PMNs). We tested the chemiluminescence response to Y. enterocolitica mutants affected in the known pYV-encoded factors. We did not detect any influence of the Yops in this phenomenon. By contrast, the presence of YadA correlated with a lack of chemiluminescence. The expression of YadA at the bacterial surface also reduced the phagocytosis by PMNs. Finally, we measured the survival of Y. enterocolitica cells confronted with PMNs by the classical plating method and by a new luminometry assay. We observed that YadA+ bacteria were not killed, while YadA- bacteria were killed. We conclude that the presence of YadA at the surface of Y. enterocolitica cells prevents phagocytosis and killing by PMNs. This conclusion is in good agreement with our recent observation that YadA protects Y. enterocolitica from opsonization by C3b.

Contrasting effects of Mycoplasma fermentans and M. felis on the viability and chemiluminescence response of human polymorphonuclear leukocytes

Contrasting effects of Mycoplasma fermentans and M. felis on the viability and chemiluminescence response of human polymorphonuclear leukocytes

The chemoluminescence response of human polymorphonuclear leukocytes to Escherichia coli O and K antigens.

The interactions between Escherichia coli O or K antigens and polymorphonuclear leukocyte function were studied. Five types of O antigen and three types of K antigen were extracted from E. coli. These included O1, O6, O75 and K1 antigens from pyelonephritopathogenic strains, O44 and K74 antigens from an enteropathogenic strain and O14 and K7 antigen from a standard strain. The antigens all reacted specifically to their specific antisera and no cross-reactions were observed. The O1 or O44 antigen stimulated a significantly greater chemoluminescence response in polymorphonuclear leukocytes obtained from normal volunteers than O75, O6 or O14 antigen. In addition, the K1 or K74 antigen stimulated polymorphonuclear leukocytes significantly more than K7 antigen. These results suggest that pyelonephritopathogenic or enteropathogenic E. coli may produce severe tissue damage as a result of the response to their O or K antigens, as well as via adhesive agents such as pyelonephritopathogenic P-pili or the enteroadhesive factor, and exotoxins such as hemolysin or verotoxin.

Effect of recombinant human interleukin-2 on the chemotaxis and chemiluminescence of human polymorphonuclear leukocytes.

Recently, unusual frequency of bacteremia and neutrophil dysfunction during interleukin-2 (IL-2) therapy for advanced cancer has been reported. To determine the cause of this dysfunction, we investigated the effects of recombinant human IL-2 on the chemotaxis and chemiluminescence (CL) response of human polymorphonuclear leukocytes (PMNs) and the effects of tumor necrosis factor-alpha (TNF-alpha), which is elevated after IL-2 administration, on the chemotaxis of human PMNs in vitro. After incubation of various concentrations of IL-2 or TNF-alpha with PMNs obtained from healthy adults, the chemotactic response to stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine was measured by a modified Boyden chamber method. The CL response of PMNs and whole blood was measured by stimulation with non-opsonized zymosan, Staphylococcus aureus or phorbol myristate acetate after 10-minute incubation with IL-2. PMN chemotaxis (IL-2 concentration: 1, 10, 100, 1000 U/ml) and the CL response of PMNs and whole blood (IL-2 concentration: 1, 10, 100 U/ml) were unchanged when compared with the responses of untreated cells. However, PMN chemotaxis was significantly inhibited after incubation with TNF-alpha at concentrations of 1, 10 and 100 U/ml. These results demonstrate that IL-2 has no direct effect on PMN function; however, TNF-alpha which increases in concentration following IL-2 therapy inhibits PMN chemotaxis, indicating that this factor may be a cause of chemotactic defects in PMNs during IL-2 therapy.

Effect of recombinant granulocyte colony-stimulating factor on the chemiluminescence response of human polymorphonuclear leukocytes

Effect of recombinant granulocyte colony-stimulating factor on the chemiluminescence response of human polymorphonuclear leukocytes

Chemiluminescence Response of Human Polymorphonuclear Leukocytes Induced by Purified, Latex Attached Klebsiella Fimbriae

Type 1 fimbriae (T1F) and type 3 fimbriae (T3F) were isolated from Klebsiella species, purified, attached to latex beads and tested for their ability to stimulate human polymorphonuclear leukocyte (PMNL) oxidative activity. The luminol dependent chemiluminescence assay was used to evaluate the response of phagocytes. Latex particles coated with type 3 fimbriae (1-T3F) induced a significantly higher chemiluminescence response than those with type 1 fimbriae (1-T1F). Opsonization of 1-T1F with pooled human serum induced chemiluminescence responses which were statistically significantly enhanced as compared to opsonized 1-T3F and both kinds of non-opsonized fimbriae.

Concurrent lipopolysaccharide enhances chemotactic response of human polymorphonuclear leukocytes to bacterial chemotaxin.

Polymorphonuclear neutrophil (PMN) function is thought to be critical in resistance to infectious agents and this implies that the PMN must be able to migrate into, and to function in, environments that may have high levels of bacterial lipopolysaccharide (LPS). Therefore, we have evaluated the effect of LPS on the in vitro migration of PMNs. Our data reveal that the human PMN is resistant to the deleterious effects of high levels of LPS, that in high concentrations LPS is, itself, a direct chemoattractant for PMNs, and that PMN migration toward a bacterial chemotaxin is enhanced if LPS is also present. Such capabilities suggest that the PMN may be uniquely qualified to migrate into microenvironments that are rich in LPS.

O-413 - Effects of New Quinolone Antibacterial Agents on Responses of Human Polymorphonuclear Leukocytes (PMNs).

O-413 - Effects of New Quinolone Antibacterial Agents on Responses of Human Polymorphonuclear Leukocytes (PMNs).

Leishmania major: inhibition of the chemiluminescent response of human polymorphonuclear leukocytes by promastigotes and their excreted factors

The effect of various leishmanial preparations on the chemiluminescent response (CR) of human polymorphonuclear leukocytes (PMN) was studied. Almost no CR was observed with PMN stimulated with either Leishmania promastigotes or their excreted factors (EFs). Promastigotes added to PMN stimulated with phorbol myristate acetate (PMA), at a proportion of 20 to 1, respectively inhibited approximately 80-83% of the CR activity. Leishmanial promastigotes, whether live or dead, infective or non-infective as well as whole parasite homogenates, soluble and insoluble fractions, all decreased the CR of the PMN (PMN-CR). A similar effect was also observed with leishmanial EFs. The degree of inhibition was dose dependent and increasing the amount of EFs in the reaction mixture resulted in decrease of the PMN-CR. More than 80% of the total activity of the PMN-CR was inhibited by 500 micrograms EFs added at zero time to the reaction mixture. The inhibition of PMN-CR obtained from a patient suffering from a chronic infection caused by L. major was not significantly different from that observed with PMN from normal donors. The present study suggests that the PMN-CR is impaired by the Leishmania parasites and their EFs and thus may allow a greater survival of the parasites within these cells.

Magnesium‐dependent induction of phagocytosis‐associated chemiluminescence of adherent human polymorphonuclear leukocytes by non‐opsonized zymosan

A severe dysfunction in the cellular response of human polymorphonuclear leukocytes (PMNL) to non-opsonized zymosan was observed under a deficiency of extracellular Mg2+. The phagocytosis-association native (luminol-independent) luminescence (NL), as well as luminol-dependent luminescence (LDL) (detected simultaneously and discriminated by spectral methods), was strongly inhibited. Apart from a general decrease of total light production, a Mg2+-concentration-dependent delay of the maximum of NL and LDL was observed. A disorder in recruitment of activated membrane-bound NADPH-oxidase of PMNL is suggested. The presence of extracellular Ca2+ did not compensate for the Mg2+ deficit. In the presence of Mg2+ only a slight Ca2+-dependent reduction of NL was obtained, but Ca2+ seemed to selectively promote LDL. This may indicate a positive influence of Ca2+ on the myeloperoxidase release from the cells. Experiments with the metalions-chelating agents EDTA and EGTA, which complex Mg2+ to differing extents, confirmed the important role of Mg2+ in PMNL-activation by non-opsonized zymosan.