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PubMed1987

Monocyte chemotaxis: a method for routine in vitro investigation using lactic dehydrogenase as a marker enzyme.

Using the cytoplasmic enzyme lactic dehydrogenase (LDH) as a marker, a method for routine measurements of highly purified monocyte chemotaxis is described. Standardization of this assay was carried out in order to achieve objective and reproducible results. The LDH-method showed a good correlation with both the 51Cr-chemotactic assay and the leading-front technique. The sensitivity of the LDH-method could be compared with the 51Cr-assay and was significantly higher compared to the leading-front technique. Being objective, inexpensive and requiring only a photometer as equipment, the LDH-method was proved to be suitable for routine investigations of monocyte chemotaxis as a part of monocyte functional activity in laboratories engaged in leukocyte research.

Cell Separation↗
PubMed1996

Neutrophil transmigration across monolayers of endothelial cells and airway epithelial cells is regulated by different mechanisms.

Neutrophil recruitment from the blood stream into the airways is one of the important features of many inflammatory disorders in the lung. In this process, neutrophils migrate first from the blood across the ECs in the apical-to-basolateral direction into the tissues and then across airway epithelium in the basolateral-to-apical direction into the lung lumen. We investigated and compared the different mechanisms of neutrophil transmigration in vitro across monolayers of these two cell types in both directions. Neutrophil migration induced by chemoattractants was stronger across resting EC than across resting epithelial cells when both cell types were growing on top of the filters (i.e., migration in the apical-to-basolateral direction). Much higher neutrophil transepithelial migration was found in the physiological, basolateral-to-apical direction (cells hanging underneath the filters) than in the opposite direction across either resting or IL-1 beta-activated epithelial cells. In contrast, no significant difference was observed with EC under these conditions. After a 4-h IL-1 beta activation, neutrophil migration across EC was dependent on IL-8 and PAF. However, the migration across epithelial cells relied, to a greater extent, on IL-8 production, and not on PAF. The adhesion molecule ICAM-1 contributed much less to neutrophil migration across epithelium than that across endothelium. Our study provides evidence of different mechanisms of neutrophil transmigration across monolayers of EC and epithelial cells in vitro, indicating that different processes control neutrophil transmigration across EC and epithelial cells in vivo.

Bronchi↗
PubMed1995

Inactivation of cell surface receptors by pheophorbide a, a green pigment isolated from Psychotria acuminata.

The inhibition of cytokine and monoclonal antibody binding cell surfaces caused by an extract of Psychotria acuminata, a medicinal plant used in the traditional medicine of the people of Belize (Central Africa), was attributed to the presence of pheophorbide a and pyropheophorbide a. Since the binding of tumor necrosis factor-alpha, interleukin-8, complement factor 5a as well as epidermal growth factor to target cells was dramatically reduced, the inhibition was not receptor or cytokine specific. In addition, the respective binding of radiolabeled monoclonal antibodies CL203 and R15.7 to the cell surface antigens intracellular cell adhesion molecule-1 and lymphocyte function-associated antigen-1 beta-chain was decreased by pretreatment of cells with pheophorbide a as well. In all cases, the inhibition by pheophorbides was dependent on the simultaneous presence of light, indicating causative involvement of a photodynamic process. These observations are not unique to pheophorbides and can be extended to porphyrins as well as to other photodynamic agents. Cytotoxicity resulting from photodynamic therapy (PDT) has been documented by many studies. Our investigations suggest that the inactivation of cell surface receptors contributes not only to an antitumor effect of PDT but also to the systemic immunosuppression, a serious side effect of PDT.

Belize↗
PubMed1989

Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors.

The neutrophil Mac-1 and gp100MEL-14 adhesion proteins are involved in neutrophil extravasation during inflammation. Both the expression and activity of Mac-1 are greatly increased after neutrophil activation. In contrast, neutrophils shed gp100MEL-14 from the cell surface within 4 minutes after activation with chemotactic factors or phorbol esters, releasing a 96-kilodalton fragment of the antigen into the supernatant. Immunohistology showed that gp100MEL-14 was downregulated on neutrophils that had extravasated into inflamed tissue. The gp100MEL-14 adhesion protein may participate in the binding of unactivated neutrophils to the endothelium; rapid shedding of gp100MEL-14 may prevent extravasation into and damage of normal tissues by activated neutrophils.

Animals↗
PubMed2000

Polarization of chemoattractant receptor signaling during neutrophil chemotaxis.

Morphologic polarity is necessary for chemotaxis of mammalian cells. As a probe of intracellular signals responsible for this asymmetry, the pleckstrin homology domain of the AKT protein kinase (or protein kinase B), tagged with the green fluorescent protein (PHAKT-GFP), was expressed in neutrophils. Upon exposure of cells to chemoattractant, PHAKT-GFP is recruited selectively to membrane at the cell's leading edge, indicating an internal signaling gradient that is much steeper than that of the chemoattractant. Translocation of PHAKT-GFP is inhibited by toxin-B from Clostridium difficile, indicating that it requires activity of one or more Rho guanosine triphosphatases.

Actins↗
PubMed2003

Shikonin, a component of chinese herbal medicine, inhibits chemokine receptor function and suppresses human immunodeficiency virus type 1.

Shikonin is a major component of zicao (purple gromwell, the dried root of Lithospermum erythrorhizon), a Chinese herbal medicine with various biological activities, including inhibition of human immunodeficiency virus (HIV) type 1 (HIV-1). G protein-coupled chemokine receptors are used by HIV-1 as coreceptors to enter the host cells. In this study, we assessed the effects of shikonin on chemokine receptor function and HIV-1 replication. The results showed that, at nanomolar concentrations, shikonin inhibited monocyte chemotaxis and calcium flux in response to a variety of CC chemokines (CCL2 [monocyte chemoattractant protein 1], CCL3 [macrophage inflammatory protein 1alpha], and CCL5 [regulated upon activation, normal T-cell expressed and secreted protein]), the CXC chemokine (CXCL12 [stromal cell-derived factor 1alpha]), and classic chemoattractants (formylmethionyl-leucine-phenylalanine and complement fraction C5a). Shikonin down-regulated surface expression of CCR5, a primary HIV-1 coreceptor, on macrophages to a greater degree than the other receptors (CCR1, CCR2, CXCR4, and the formyl peptide receptor) did. CCR5 mRNA expression was also down-regulated by the compound. Additionally, shikonin inhibited the replication of a multidrug-resistant strain and pediatric clinical isolates of HIV in human peripheral blood mononuclear cells, with 50% inhibitory concentrations (IC(50)s) ranging from 96 to 366 nM. Shikonin also effectively inhibited the replication of the HIV Ba-L isolate in monocytes/macrophages, with an IC(50) of 470 nM. Our results suggest that the anti-HIV and anti-inflammatory activities of shikonin may be related to its interference with chemokine receptor expression and function. Therefore, shikonin, as a naturally occurring, low-molecular-weight pan-chemokine receptor inhibitor, constitutes a basis for the development of novel anti-HIV therapeutic agents.

Anti-HIV Agents↗
PubMed2000

Pathogenesis of group A streptococcal infections.

Group A streptococci are model extracellular gram-positive pathogens responsible for pharyngitis, impetigo, rheumatic fever, and acute glomerulonephritis. A resurgence of invasive streptococcal diseases and rheumatic fever has appeared in outbreaks over the past 10 years, with a predominant M1 serotype as well as others identified with the outbreaks. emm (M protein) gene sequencing has changed serotyping, and new virulence genes and new virulence regulatory networks have been defined. The emm gene superfamily has expanded to include antiphagocytic molecules and immunoglobulin-binding proteins with common structural features. At least nine superantigens have been characterized, all of which may contribute to toxic streptococcal syndrome. An emerging theme is the dichotomy between skin and throat strains in their epidemiology and genetic makeup. Eleven adhesins have been reported, and surface plasmin-binding proteins have been defined. The strong resistance of the group A streptococcus to phagocytosis is related to factor H and fibrinogen binding by M protein and to disarming complement component C5a by the C5a peptidase. Molecular mimicry appears to play a role in autoimmune mechanisms involved in rheumatic fever, while nephritis strain-associated proteins may lead to immune-mediated acute glomerulonephritis. Vaccine strategies have focused on recombinant M protein and C5a peptidase vaccines, and mucosal vaccine delivery systems are under investigation.

Amino Acid Sequence↗
PubMed1982

Chemotaxis of purified human monocytes in vitro: lack of accessory cell requirement.

This study was undertaken to determine whether cell cooperation, either among monocytes or between monocytes and lymphocytes, is a prerequisite for monocyte chemotactic responsiveness. We compared Ficoll-Hypaque-separated mononuclear cells and a preparation of 99% pure monocytes obtained by chemotaxis in a newly designed separation chamber. Monocytes of both preparations migrated to chemoattractants without a lag phase, and no further increase in migrated cells was observed after 70 min. The cell dose-response was linear for both preparations over a wide range of cell concentrations in the cell input well of the chemotaxis chamber, suggesting that no monocyte-monocyte interaction was required. Since only 20 to 60% of the monocytes purified by chemotaxis migrated a second time, the possibility of a requirement for an accessory cell was tested. The addition to purified monocytes of several different mononuclear cell preparations comprising lymphocytes or nonmigrating monocytes had no effect on monocyte migration. These experiments show that normal human blood monocytes in vitro do not require stimuli from other cells to respond to chemoattractants. Their behavior is profoundly different from that of mouse peritoneal macrophages, which exhibit a time lag in vitro before migration toward an attractant and become more responsive with either increasing cell concentration or addition of purified lymphocytes.

Cell Communication↗
PubMed1984

Interaction of group A streptococcal peptidoglycan polysaccharide with human polymorphonuclear leukocytes: implications for pathogenesis of chronic inflammation.

Injection of sterile aqueous preparations of the peptidoglycan polysaccharide of group A streptococci produces chronic inflammation in several animal models. Accordingly, the effect of peptidoglycan and group A-specific polysaccharide (PG-APS) polymers on human polymorphonuclear neutrophil oxidative metabolism was studied with the supposition that this interaction may contribute to the inflammation observed. PG-APS in concentrations of 1.0 to 100 micrograms/ml stimulated oxygen consumption and hexose monophosphate shunt activity in the presence of 10% normal serum in a dose-related manner. Stimulation did not occur in serum-free media and was reduced in media with heat-treated serum. The stimulation of hexose monophosphate shunt activity by PG-APS opsonized with normal serum (bound complement components) and the activated supernatant from which PG-APS had been removed by centrifugation (presumably containing the soluble complement component, C5a) demonstrated 79 and 75%, respectively, of the stimulation with PG-APS in the presence of 10% normal serum. PG-APS triggered release of O2- (3.4 +/- 2.0 nmol by 10(6) human polymorphonuclear neutrophils over 30 min), which was significantly enhanced (9.6 +/- 2.9 nmol O2-) by treatment of cells with cytochalasin B. These results show that PG-APS interacts with serum in such a fashion as to activate human polymorphonuclear neutrophil metabolism and increase secretion of O2-.

Blood Glucose↗
PubMed1992

Preliminary characterization of Pseudomonas aeruginosa peptide chemotactins for polymorphonuclear leukocytes.

In a previous report, we showed that supernatants of Pseudomonas aeruginosa cultures exhibit chemotactic activity for polymorphonuclear leukocytes (PMNL). In this study, P. aeruginosa chemotactins were isolated, purified, and partially characterized. The organisms were cultured in Vogel-Bonner defined medium, and cultures were stopped in late log phase. Chemotactins withstood heating, remained unaltered after acid or alkali treatment in a pH range from 4 to 10, and resisted digestion by trypsin or carboxypeptidase, but chemotactic activity was decreased by 73% after incubation with pronase. Only 2% of the total chemotactic activity of culture supernatants could be extracted with chloroform. Chemotactins with molecular sizes less than 3 kDa constituted the largest contribution to the chemotactic activity of culture supernatants. Pretreatment of PMNL with 10(-5) M formylmethionyl-leucyl-phenylalanine (FMLP) inhibited chemotaxis towards FMLP and P. aeruginosa culture supernatants but not towards complement component C5a. In conclusion, the total chemotactic activity for PMNL of P. aeruginosa culture supernatants was due, almost exclusively, to chemotactins that have properties similar, if not identical, to those exhibited by formylmethionyl peptides.

Bacterial Proteins↗
PubMed2006

Expression microarray and mouse virulence analysis of four conserved two-component gene regulatory systems in group a streptococcus.

Group A streptococcus (GAS) is a gram-positive human bacterial pathogen that causes diseases ranging from relatively mild epithelial cell surface infections to life-threatening invasive episodes. Much is known about the extracellular molecules that contribute to host-pathogen interactions, but in contrast, far less information is available about regulatory genes that control the expression of individual or multiple GAS virulence factors. The eight GAS genomes that have been sequenced have 12 conserved two-component gene regulatory systems (TCSs), but only 3 of these 12 have been studied in detail. Using an allelic replacement strategy with a nonpolar cassette, we inactivated the response regulator of four TCSs that have only weak homology with TCS genes of known or inferred function in other bacteria. The mutant strains were analyzed by expression microarray analysis at four time points and tested in two mouse infection models. Each TCS influenced expression (directly or indirectly) of 12 to 41% of all chromosomal genes, as assessed by growth in Todd-Hewitt broth and a custom Affymetrix GeneChip. None of the isogenic mutant strains was significantly altered for mouse virulence based on intraperitoneal inoculation. Similarly, compared to the wild-type strain, there was no significant difference in skin lesion size for three of the four mutants. In contrast, the DeltaM5005_Spy_0680 mutant strain produced significantly larger abscesses after subcutaneous inoculation into mice, consistent with a hypervirulence phenotype. The mutant strain had significantly higher in vitro expression of several proven and putative virulence genes, including scpA, encoding a peptidase that inactivates complement protein C5a. Together, the data provide new information about previously uncharacterized GAS TCSs.

Animals↗
PubMed1988

Complement activation and HLA-B27.

The efficiency of complement activation was studied in sera from HLA-B27 positive and negative subjects (27 with previous yersinia arthritis and 35 controls). Activation of complement with zymosan induced higher mean levels of the anaphylatoxin C3a in HLA-B27 positive sera (mean (SD) 7.40 (1.66) mg/l) than in HLA-B27 negative sera (6.41 (1.79) mg/l). Similarly, higher levels of C3d,g, another C3 breakdown fragment, were obtained in HLA-B27 positive sera after Escherichia coli 0111:B4 lipopolysaccharide treatment (17.6 (3.7)% v 15.0 (3.8)%). The differences occurred irrespective of previous arthritis, complement C4 or Bf phenotype, or variation in background complement levels. The findings suggest that an increased responsiveness to complement activators may contribute to the pathogenesis of HLA-B27 associated inflammatory diseases.

Adult↗