Biomedical subjects
Publications and source records attributed to A Kharazmi.
The interaction of human neutrophils with Pseudomonas aeruginosa biofilms was examined by using a chemiluminescence assay. The biofilms induced an oxidative burst response by polymorphonuclear leukocytes which was slow and only 25% of the response to planktonic bacteria. The reduced response to P. aeruginosa biofilms could play a role in the persistence of bacteria in chronic infections.
Adherence of P. aeruginosa to the lining of the respiratory tract is probably mediated by interaction of pili or alginate with lactosyl and sialosyl residues on the respiratory cells. The toxins produced by P. aeruginosa (for example, elastase and alkaline protease) may play a key role during the initial persistent colonization as they interfere with important defense mechanisms. Neutralizing antibodies are eventually produced, and a poor prognosis is correlated to a pronounced antibody response. The bacteria are protected against the host's defense mechanisms by production of alginate which encapsulates microcolonies of P. aeruginosa in the lungs. During the chronic infection, toxins of P. aeruginosa probably play little if any direct pathogenic role, however, immune complexes seem to be a major trigger of chronic inflammation in the lungs. Proteolytic enzymes and oxygen radicals released from the abundance of neutrophils in the lungs are probably responsible for most of the tissue damage. The individual course of the chronic infection may be explained by regulatory mechanisms, such as cleavage of immune complexes by neutrophil elastase, and by the balance between the different IgG subclass-specific antibody responses.
The ability of peptidoglycan from Staphylococcus epidermidis and Staphylococcus aureus to generate in human serum chemoattractant for peripheral blood neutrophils was studied. It was shown that PG from the two bacteria was able to induce chemotactic activity in normal human serum. Sonication of PG was required to generate this activity. Very little or no activity was generated in heat-treated or C5-deficient human serum by PG, indicating that PG treatment of serum resulted in generation of chemoattractants by activation of complement. Kinetics studies employing C2-deficient or MgEGTA-chelated serum revealed that S. epidermidis induced chemotactic activity by activating the alternative complement pathway. The alternative complement activation induced by S. epidermidis occurred rapidly and was completed after 15 min, whereas S. aureus activated the alternative pathway much more slowly, with activation reaching a maximum at 60 min. The rapid activation of the alternative complement pathway by S. epidermidis PG may partly explain why this bacterium does not normally cause infections in healthy individuals.
Interleukin 6 (IL-6), a 26-kDa inducible protein, is a cytokine with multiple biological activities. This paper reports on the regulatory role of rIL-6 on the function of human polymorphonuclear and mononuclear leukocytes, a property not described previously. rIL-6 by itself did not exhibit any chemotactic activity and it could not activate these cells for an oxidative burst response. Preincubation of both cell types with rIL-6 at concentrations of 5 and 50 ng/ml primed the cells for enhanced generation of oxygen radicals following stimulation with the chemotactic peptide f-Met-Leu-Phe or the phorbol ester PMA. The enhancement of the oxidative burst response occurred both at the level of superoxide anion generation, an early step in the activation pathway, and at the level of the hydrogen peroxide-myeloperoxidase mediated response, a later step in the oxidative burst pathway. The priming ability was abolished by heat treatment of rIL-6 at 100 degrees C but not at 70 degrees C. Stimulation of B cell growth and immunoglobulin production combined with enhancement of oxidative burst response of phagocytic cells by IL-6 provide an effective mechanism of fighting against invading micro-organisms.
Modulation of the oxidative burst responsiveness of human blood monocytes and neutrophils after incubation with purified exotoxin A from Pseudomonas aeruginosa was studied in a lucigenin- and luminol-enhanced chemiluminescence system. Exotoxin A alone caused a dose-dependent stimulation of monocyte chemiluminescence responses, whereas neutrophil responses were inconsistent. Preincubation of monocytes with exotoxin A primed the cells for a significantly higher oxidative burst response when N-f-methionyl-leucyl-phenylalanine (fMLP) was used as a secondary stimulus, especially in the lucigenin-enhanced system. Heat-treatment at 100 degrees C for 15 min completely abolished the priming activity of the exotoxin A preparation. These findings demonstrate that exotoxin A modulates monocyte responsiveness in the chemiluminescence assay and suggest that increased release of toxic oxygen radicals from mononuclear phagocytes may contribute to the tissue damage in lungs with chronic P. aeruginosa infections.
Infection of host cells with Leishmania, which are obligate parasites of mononuclear phagocytes, most probably involved chemotaxis of host cells towards the parasite. We have examined the chemotactic properties of a sonicate derived from L. mexicana amazonensis promastigotes for normal human peripheral blood monocytes and neutrophils. L. m. amazonensis sonicate exhibited chemotactic activity for monocytes and neutrophils. Treatment at 65 degrees C for 30 min, enhanced the activity for neutrophils but not for monocytes, while treatment at 100 degrees C for 60 min abolished the activity. Additional studies showed that the sonicate generated chemotactic activity in serum, presumably by activating the alternative complement pathway to produce C5a, for monocytes and neutrophils. Incubation of monocytes and neutrophils with the sonicate inhibited the chemotactic activity of these cells towards various chemoattractants. When the sonicate was heat-treated the inhibitory effect was lost, except when sonicate was used as a chemoattractant. These results indicate the presence of specific receptors for factor(s) from L. m. amazonensis promastigotes on human monocytes and neutrophils.
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Plasma endotoxin concentrations and oxidative burst response of peripheral blood polymorphonuclear leukocytes were examined in 12 patients undergoing coronary artery bypass. The measurements were made just before the operation, 5 minutes after removal of the aortic crossclamp, and 24 hours after the operation. Endotoxin was quantitated by a combination of a sensitive Limulus amebocyte lysate assay and rocket immunoelectrophoresis measuring picogram amounts of endotoxin. Peripheral blood neutrophils were purified by a two-step dextran sedimentation and metrizoate sodium Ficoll (Lymphoprep., Nyegaard, Oslo, Norway) centrifugation. The oxidative burst response of these cells was measured for their ability to generate superoxide anion and was determined by a cytochrome c reduction assay. Preoperatively, all the plasma samples except one were free of endotoxin. The endotoxin levels reached 100 pg/ml 5 minutes after removal of the aortic crossclamp, and except in one sample they had decreased 24 hours after the operation. Studies on the generation of superoxide by neutrophils showed a decline in the response 5 minutes after removal of the aortic crossclamp and an enhancement of the response to f-Met-Leu-Phe by cells obtained from 11 of 12 patients 24 hours postoperatively. In vitro addition of bacterial lipopolysaccharide to blood from healthy individuals also enhanced the superoxide response of the neutrophils. We conclude that during cardiopulmonary bypass the circulating blood is contaminated by endotoxin and the neutrophils are primed for enhanced generation of oxygen radicals. The released oxygen radicals may be involved in the tissue damage observed in these patients.
The effect of N-acetylcysteine (NAC) on human neutrophil and monocyte cell viability, chemotaxis, oxygen consumption and chemiluminescence was studied. It was found that NAC at concentrations higher than 3 X 10(-2) M resulted in neutrophil and monocyte cytotoxicity. The studies on the effect of NAC on neutrophil and monocyte chemotaxis showed that NAC inhibited chemotaxis of both cell types in a concentration dependent manner. NAC at 3 X 10(-2) M inhibited chemotaxis of both cell types by about 50% and at 10(-1) M inhibited PMN chemotaxis by 95% and MNL chemotaxis by 85%. The studies on the effect of NAC on neutrophil chemiluminescence demonstrated that NAC at concentrations of 1.5 X 10(-2) M, or higher, inhibited the response of the activated cells totally. When pH adjusted NAC or Mucomyst was used the inhibition was observed with higher concentrations of the drug (1.5 X 10(-1) M). NAC exhibited a similar pattern of inhibition on monocyte chemiluminescence response. These findings demonstrate that NAC, at concentrations obtainable in vivo by inhalation, impairs the chemotaxis and generation of oxygen radicals by human phagocytic cells. This property of NAC could have important implications concerning the prevention of tissue damage caused by these cells in inflammatory areas.
The effects of recombinant TNF and GM-CSF on human peripheral blood neutrophil and monocyte chemotaxis and the superoxide response were studied. TNF exhibited a slight chemotactic activity for both cell types. Preincubation of neutrophils with as little as 40 units/ml strongly inhibited the neutrophil chemotaxis towards f-Met-Leu-Phe. The inhibition of monocyte chemotaxis required higher concentrations of TNF (greater than 400 units/ml). TNF at concentrations higher than 500 units/ml enhanced the generation of superoxide anions by neutrophils stimulated with f-Met-Leu-Phe. In contrast, TNF even at 2,000 units/ml did not prime monocytes for enhanced superoxide response. GM-CSF alone did not exhibit any chemotactic activity for any of the cell types tested. Preincubation of cells with GM-CSF inhibited chemotaxis of neutrophils but not of monocytes. GM-CSF was as potent as TNF in enhancing the generation of superoxide response by neutrophils. However, GM-CSF did not have any effect on monocyte superoxide response. The priming ability of TNF and GM-CSF on neutrophils was heat-sensitive. We conclude that TNF and GM-CSF play a more pronounced regulatory role on neutrophils than on monocytes. Inhibition of neutrophil chemotaxis followed by enhancement of the superoxide response by TNF and GM-CSF may provide an attractive mechanism by which these cytokines assist in fighting invading microorganisms.
We report that recombinant human IL 1 alpha and beta and the two synthetic short fragments 163-171 and 226-254 do not exhibit chemotactic activity for human peripheral blood neutrophils and monocytes. These products up to concentrations of 4 X 10(4) units/ml failed to show chemotactic activity. Furthermore, IL 1 alpha and beta failed to generate chemotactic factors from human serum. Recombinant IL 1 alpha, IL 1 beta, or IL 1 beta fragments 163-171 and 226-254 did not induce any superoxide response by monocytes or neutrophils. However, exposure of monocytes to recombinant IL 1 alpha or IL 1 beta resulted in enhanced generation of superoxide response following stimulation with PMA. No priming was observed in neutrophils. These results suggest that IL 1 alpha and beta are involved in regulation of monocyte oxidative burst response, but play no direct regulatory role on neutrophil function.
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Blood mononuclear cells (PBMC) recognizing soluble malaria antigens (SPag) are present in the peripheral blood of individuals clinically immune to malaria, and they proliferate after exposure to such antigens. To test whether these cells have effector activity against Plasmodium falciparum, we stimulated PBMC from malaria-immune donors by SPag and purified protein derivative (PPD) in culture for 7 days. The PBMC were then co-incubated with P. falciparum for 48 h, and parasitaemia was determined by microscopy. Parasite growth was only significantly impaired after incubation with PBMC stimulated by either SPag or PPD in the presence of immune serum. Studies on subpopulations of PBMC indicated that the inhibitory cells resided among the adherent cell fraction. Furthermore we tested PBMC for cytotoxic activity against P. falciparum-infected autologous or heterologous erythrocytes. Experiments were done both in the absence and the presence of immune serum. Neither fresh PBMC nor PBMC activated by SPag or PPD for 7 days prior to assay were cytotoxic, indicating that cytotoxic T cells, natural killer (NK) cells, and K cells did not possess cytotoxic activity directed against parasitized erythrocytes. These data support the hypothesis that activated monocytes are the most important effector cells in the peripheral blood of malaria immune individuals.
It has been recently reported that neutrophils are involved in the regulation of NK cell activity. However, the mechanism of such regulation is unclear. The present study was designed to investigate the mechanisms involved in the regulation of NK cytotoxicity by human neutrophils. The role of indomethacin, an anti-inflammatory drug, in this interaction was studied. NK cells were purified from peripheral blood obtained from normal individuals. NK cell cytotoxicity was tested on K 562 cell line by Cr release assay. Autologous neutrophils obtained from peripheral blood were stimulated by opsonized zymosan either in the presence or absence of indomethacin. The role of neutrophil supernatant containing oxygen radicals and prostaglandins on NK cytotoxicity was examined. It was shown that supernatants from stimulated neutrophils significantly inhibited (P less than 0.05) the autologous NK cell cytotoxicity. The presence of indomethacin in the in vitro reaction mixture, or given orally to donors, partially or completely abolished the inhibitory effect of neutrophil supernatant. Indomethacin inhibited prostaglandin E2 release, and luminol-enhanced, myeloperoxidase-mediated chemiluminescence of activated PMN. Diafiltration of neutrophil supernatant showed that the inhibitory activity was present in the fraction containing molecules lower than 5,000 daltons. In conclusion, our findings indicate that down-regulation of NK cytotoxicity is mediated by prostaglandins produced by stimulated neutrophils and possibly by oxygen radicals.
Actinobacillus species are usually not considered as being human pathogens apart from A. actinomycetemcomitans. However, single cases of human meningitis, septicemia, and empyema caused by Actinobacillus lignieresii have been reported in the literature. This is the first reported case of Actinobacillus hominis giving rise to pleural-empyema in a patient with carcinoma of the lung. The function of peripheral blood neutrophils, serum opsonic activity and specific precipitating antibodies were investigated. Neutrophils from the patient exhibited an enhanced oxidative burst response measured by chemiluminescence assay. Furthermore, the opsonic activity of the serum from the patient was higher than that of a healthy control person. Several precipitating antibodies to various antigens of Actinobacillus hominis were demonstrated in the serum of the patient by crossed immunoelectrophoresis.
N-acetylcysteine (NAC) is known to be a scavenger of free oxygen radicals, and recent in vitro studies have demonstrated that it is also able to inhibit leukocyte function. The clinical significance of these effects is, however, not known. In this study we have measured the effect on human blood neutrophil and monocyte function of a single 400 mg dose of NAC administered orally. Administration of NAC to ten healthy volunteers resulted in significant reduction of neutrophil chemiluminescence response following activation by opsonized zymosan as compared to four non-treated persons acting as controls. No effect was observed on the chemotaxis of either cell type or on monocyte chemiluminescence response. These findings suggest that NAC may be beneficial in clinical conditions like cystic fibrosis, where tissue damage may be a consequence of the effects of increased release of toxic oxygen radicals and proteolytic enzymes.
This study was undertaken to determine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (ELA) on human lymphocyte function. AP at 50 micrograms/ml and ELA at 12 micrograms/ml caused a 50% inhibition of phytohemagglutinin-induced proliferation. There was no difference in the effect of proteases on CD4- and CD8-positive cells. To determine the effect of proteases on interleukin-2 (IL-2)-induced cell proliferation, the proteases and IL-2 were added to the IL-2-dependent CTLL-2 cell line. AP and ELA inhibited the proliferation of these cells. When IL-2 was added in excess, the inhibition was partly reversed. ELA at 10 micrograms/ml cleaved IL-2, as judged by size chromatography of a reaction mixture containing 125I-labeled IL-2 and the proteases. The ELA-digested IL-2 exhibited a reduced binding capacity to IL-2 receptors on the lymphocytes. Furthermore, treatment of phytohemagglutinin-stimulated lymphocytes with AP and ELA resulted in inhibition of binding of intact IL-2 to IL-2 receptors on the stimulated lymphocytes. These results indicated that P. aeruginosa-derived enzymes are able to interfere with human lymphocyte function in vitro and that this effect might be due to cleavage of IL-2.
The spontaneous binding of both piliated and non-piliated E. coli to human lymphocytes, monocytes, and polymorphonuclear leukocytes (PMN) was investigated. It was found that the piliated E. coli showed a higher binding percentage to lymphocytes than the nonpiliated bacteria. A specific antiserum raised in rabbit against E. coli strongly inhibited the binding of the homologous E. coli strain to lymphocytes and monocytes. On the other hand there was no difference in the binding of piliated and non-piliated E. coli to PMNs, and the specific E. coli antiserum did not inhibit the binding. The binding of E. coli to human lymphocytes obtained from healthy donors was investigated in a 3-week follow-up experiment. It was shown that there were significant differences between the binding of cells from different individuals, whereas there were no differences in the binding ability of the cells from the same individual in the 3-week follow up.