Torbjörn Bengtsson

Activation of the granule pool of the NADPH oxidase accelerates apoptosis in human neutrophils

Oxidative stress induces apoptosis in many types of cells, including human neutrophils. Our objective was to determine whether reactive oxygen species (ROS) produced by activated neutrophils are associated with accelerated apoptosis. Exposing neutrophils to ionomycin or phorbol myristate acetate (PMA) induced intracellular H2O2 production and rapid onset of apoptosis, measured as condensed chromatin, cellular shrinkage, and DNA fragmentation. Neutrophils activated with formyl‐methionyl‐leucyl‐phenylalanine (fMLP) generated mainly extracellular H2O2 and did not undergo apoptosis. Exogenously added H2O2, together with the catalase blocker sodium azide, induced apoptosis to the same extent and with similar kinetics as PMA and ionomycin. Adenosine inhibited ionomycin‐induced intracellular H2O2 production and apoptosis. Neither PMA nor ionomycin caused apoptosis in dimethyl sulfoxide‐differentiated HL‐60 cells, which are incapable of intracellular H2O2 production, whereas H2O2 induced apoptosis more efficiently in these cells than in neutrophils. We propose that activated neutrophils use intracellularly formed H2O2 to commit suicide. J. Leukoc. Biol. 65: 196–204; 1999.

Synthesis and Characterization of PEGylated Gd2O3 Nanoparticles for MRI Contrast Enhancement

Recently, much attention has been given to the development of biofunctionalized nanoparticles with magnetic properties for novel biomedical imaging. Guided, smart, targeting nanoparticulate magnetic resonance imaging (MRI) contrast agents inducing high MRI signal will be valuable tools for future tissue specific imaging and investigation of molecular and cellular events. In this study, we report a new design of functionalized ultrasmall rare earth based nanoparticles to be used as a positive contrast agent in MRI. The relaxivity is compared to commercially available Gd based chelates. The synthesis, PEGylation, and dialysis of small (3-5 nm) gadolinium oxide (DEG-Gd(2)O(3)) nanoparticles are presented. The chemical and physical properties of the nanomaterial were investigated with Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, and dynamic light scattering. Neutrophil activation after exposure to this nanomaterial was studied by means of fluorescence microscopy. The proton relaxation times as a function of dialysis time and functionalization were measured at 1.5 T. A capping procedure introducing stabilizing properties was designed and verified, and the dialysis effects were evaluated. A higher proton relaxivity was obtained for as-synthesized diethylene glycol (DEG)-Gd(2)O(3) nanoparticles compared to commercial Gd-DTPA. A slight decrease of the relaxivity for as-synthesized DEG-Gd(2)O(3) nanoparticles as a function of dialysis time was observed. The results for functionalized nanoparticles showed a considerable relaxivity increase for particles dialyzed extensively with r(1) and r(2) values approximately 4 times the corresponding values for Gd-DTPA. The microscopy study showed that PEGylated nanoparticles do not activate neutrophils in contrast to uncapped Gd(2)O(3). Finally, the nanoparticles are equipped with Rhodamine to show that our PEGylated nanoparticles are available for further coupling chemistry, and thus prepared for targeting purposes. The long term goal is to design a powerful, directed contrast agent for MRI examinations with specific targeting possibilities and with properties inducing local contrast, that is, an extremely high MR signal at the cellular and molecular level.

Gingipains from the Periodontal Pathogen Porphyromonas gingivalis Play a Significant Role in Regulation of Angiopoietin 1 and Angiopoietin 2 in Human Aortic Smooth Muscle Cells

ABSTRACT Angiopoietin 1 (Angpt1) and angiopoietin 2 (Angpt2) are the ligands of tyrosine kinase (Tie) receptors, and they play important roles in vessel formation and the development of inflammatory diseases, such as atherosclerosis. Porphyromonas gingivalis is a Gram-negative periodontal bacterium that is thought to contribute to the progression of cardiovascular disease. The aim of this study was to investigate the role of P. gingivalis infection in the modulation of Angpt1 and Angpt2 in human aortic smooth muscle cells (AoSMCs). We exposed AoSMCs to wild-type (W50 and 381), gingipain mutant (E8 and K1A), and fimbrial mutant (DPG-3 and KRX-178) P. gingivalis strains and to different concentrations of tumor necrosis factor (TNF). The atherosclerosis risk factor TNF was used as a positive control in this study. We found that P. gingivalis (wild type, K1A, DPG3, and KRX178) and TNF upregulated the expression of Angpt2 and its transcription factor ETS1, respectively, in AoSMCs. In contrast, Angpt1 was inhibited by P. gingivalis and TNF. However, the RgpAB mutant E8 had no effect on the expression of Angpt1, Angpt2, or ETS1 in AoSMCs. The results also showed that ETS1 is critical for P. gingivalis induction of Angpt2. Exposure to Angpt2 protein enhanced the migration of AoSMCs but had no effect on proliferation. This study demonstrates that gingipains are crucial to the ability of P. gingivalis to markedly increase the expressed Angpt2/Angpt1 ratio in AoSMCs, which determines the regulatory role of angiopoietins in angiogenesis and their involvement in the development of atherosclerosis. These findings further support the association between periodontitis and cardiovascular disease.

Actin assembly and regulation of neutrophil function: effects of cytochalasin B and tetracaine on chemotactic peptide-induced O2- production and degranulation.

Several studies indicate that the actin filament system is not only involved in cell motility, but also in the regulation of other neutrophil functions. The aim of the present investigation was to examine the mechanisms by which actin filament formation participates in the control of the respiratory burst and degranulation in human neutrophils. The approach taken was to use both an inhibitor (cytochalasin B) and a potentiator (tetracaine) of formylmethionyl‐leucyl‐phenylalanine (fMet‐Leu‐Phe)‐induced actin polymerization. The total inhibition of fMet‐Leu‐Phe‐induced actin polymerization in cytochalasin B‐treated cells was accompanied by an impressive potentiation of both oxidase activity and degranulation (azurophilic and specific granules). However, preincubation with tetracaine, which causes an enhanced accumulation of F‐actin in the periphery of fMet‐Leu‐Phe‐stimulated cells, also augmented the rate and duration of peptide‐induced superoxide anion production, but inhibited degranulation (specific granules). A likely explanation for the potentiating effects of cytochalasin B and tetracaine is provided by our observation that both of these substances reduced the acid‐resistant binding of fMet‐Leu‐Phe (interpreted as a decrease in the internalization of fMet‐Leu‐Phe‐receptor‐complexes), resulting in an enhanced formation of second messengers (diacylglycerol). The findings that tetracaine potentiated the activity of the oxidase, whereas it inhibited degranulation (specific granules), suggest that actin polymerization per se plays a role in the latter process. Consequently, this study argues against the idea of a direct inhibitory effect of F‐actin on chemotactic factor‐induced oxidase activation, but supports an active role of actin filaments in the translocation and release of granule components.

On the binding of complement to solid artificial surfaces in vitro.

Since the realization of a complement activation capacity by artificial surfaces upon contact with blood, a common belief has evolved that charged nucleophilic surface groups such as amine (-NH2) and hydroxyl (-OH) react with and eventually bind to the internal thioester in complement factor 3 (C3). A covalent amide or ester linkage is thereby supposed to form between C3b and the surface itself. In this report, we present complement surface binding data by null-ellipsometry for two nucleophilic surfaces (-NH2 and -OH), for surfaces with immunoglobulin G (IgG) covalently bound, and for IgG spontaneously pre-adsorbed to hydrophobic silicon. The results reveal that the plasma proteins that were deposited during complement activation became eluted by sodium dodecyl sulfate. Hence the direct covalent binding between C3 and solid nucleophilic surfaces seems to be only of moderate importance, at least during shorter serum incubations. This strongly suggests that the prevalent covalent linkage model between solid artificial surfaces and C3b is not accurate. Instead we suggest a more pronounced role for C3 associations to other adsorbed proteins and or electrostatic and hydrophobic protein-surface interactions.

Platelets enhance Fc(gamma) receptor-mediated phagocytosis and respiratory burst in neutrophils: the role of purinergic modulation and actin polymerization.

The interaction of platelets with neutrophil granulocytes is considered to play an important role in the inflammatory process, and the present study was focused on platelet‐induced modulation of Fcγy receptor‐mediated functions in neutrophils. We found that phagocytosis and the respiratory burst (measured as luminol‐enhanced chemiluminescence), triggered in neutrophils by immunoglobulin G (IgG)‐opsonized yeast particles, were potentiated by platelets and that maximal enhancement was achieved at a physiological neutrophil/platelet ratio of about 1:50 to 1:100. Platelets both increased the intra‐ and extracellular generation of oxygen radicals as well as the release of myeloperoxidase from stimulated neutrophils. The presence of platelets also induced a cortical actin polymerization in neutrophils, which might explain the increased phagocytic capacity. Platelets appear to affect neutrophil function in a contact‐independent manner that most likely involves ATP, indicated by the following: (1) platelet supernatants, but not fixed platelets, affected neutrophil function in the same way as viable platelets; (2) platelets raised the extracellular ATP level four‐ to fivefold; (3) exogenous ATP mimicked the effects of platelets on actin polymerization, phagocytosis, and the respiratory burst in neutrophils; (4) hydrolysis of extracellular ATP with apyrase or blocking of ATP receptors with suramin reversed the platelet‐induced enhancement of neutrophil function. An increased accumulation of extracellular adenosine, induced by inhibiting endogenous adenosine deaminase or adding exogenous adenosine, reversed the effects of platelets. The platelet‐induced potentiation of the respiratory burst was inhibited by the tyrosine kinase inhibitor genistein, suggesting that tyrosine phosphorylation is involved. However, platelets did not significantly affect the Fcγ receptor‐triggered calcium response in neutrophils. In conclusion, we show that platelets, through an ATP‐dependent mechanism, potentiate IgG‐mediated ingestion and production of oxygen metabolites in neutrophils.

The influence of plasma proteins and platelets on oxygen radical production and F-actin distribution in neutrophils adhering to polymer surfaces

It is well known that blood cell interactions with artificial surfaces might have deleterious effects on host tissue, however, the mechanisms involved are far from understood. In this study, neutrophil-platelet interaction on uncoated or protein-coated polymer surfaces was investigated. Cell spreading, reorganization of actin filaments and release of oxygen metabolites (measured as luminol-amplified chemiluminescence) were used as criteria for cell activation on positively charged, hydrophilic 1,2-diaminocyclohexane, and negatively charged, hydrophobic hexamethylene-disiloxane. The model surfaces were made by radio frequency plasma discharge polymerization. Neutrophil contact with the uncoated polymers induced a prolonged generation of oxygen radicals. Precoating of the polymer surfaces with human serum albumin (HSA) or fibrinogen, markedly reduced neutrophil activation, whereas coating with human immunoglobulin G (IgG), a well-known opsonin, resulted in significantly higher levels of cell activation. Consequently, protein coating overruled the activating effects of the polymer surfaces. The presence of unstimulated or thrombin-stimulated platelets markedly increased the reactivity of neutrophils against fibrinogen- and IgG-coated surfaces. However, neutrophils remained relatively unreactive in the presence of platelets on HSA-treated surfaces. Comparison of the different types of surfaces used, reveals a correlation between the degree of cell spreading, reorganization of the actin cytoskeleton and the amount of oxygen radicals produced. Our results suggest that the acute inflammatory reaction on a biomaterial surface is highly dependent on the nature and composition of the first adsorbed protein layer and the extent of platelet activation.

Lipoxin A4 Inhibits Porphyromonas gingivalis-Induced Aggregation and Reactive Oxygen Species Production by Modulating Neutrophil-Platelet Interaction and CD11b Expression

ABSTRACT Porphyromonas gingivalis is an etiological agent that is strongly associated with periodontal disease, and it correlates with numerous inflammatory disorders, such as cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. Lipoxin A4 (LXA4) is an endogenous anti-inflammatory and proresolving mediator that is protective of inflammatory disorders. The aim of this study was to investigate the effect of LXA4 on the P. gingivalis-induced activation of neutrophils and platelets and the possible involvement of Rho GTPases and CD11b/CD18 integrins. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry and fluorescence microscopy. Integrin activity was studied by flow cytometry, detecting the surface expression of CD11b/CD18 as well as the exposure of the high-affinity integrin epitope, whereas the activation of Rac2/Cdc42 was examined using a glutathione S-transferase pulldown assay. The study shows that P. gingivalis activates Rac2 and Cdc42 and upregulates CD11b/CD18 and its high-affinity epitope on neutrophils, and that these effects are diminished by LXA4. Furthermore, we found that LXA4 significantly inhibits P. gingivalis-induced aggregation and ROS generation in whole blood. However, in platelet-depleted blood and in isolated neutrophils and platelets, LXA4 was unable to inhibit either aggregation or ROS production, respectively. In conclusion, this study suggests that LXA4 antagonizes P. gingivalis-induced cell activation in a manner that is dependent on leukocyte-platelet interaction, likely via the inhibition of Rho GTPase signaling and the downregulation of CD11b/CD18. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.

Cytokines and chemokines are differentially expressed in patients with periodontitis: possible role for TGF-β1 as a marker for disease progression.

Periodontitis is a chronic inflammatory disease characterized by destruction of periodontal tissue ultimately leading to bone destruction and has been associated with other inflammatory diseases, such as atherosclerosis. Attachment loss of periodontal tissue is primarily caused by host cell-derived immune responses against subgingival biofilm. The aim of the present study was to determine the cytokine profile in serum, saliva and gingival crevicular fluid (GCF) patients with periodontitis and healthy controls. We show that periodontitis patients exhibit higher numbers of periodontal pathogens and their immune responses are significantly altered. The levels of IL-6 in saliva and GCF were significantly suppressed, and while CXCL8 was not altered in serum, its expression levels were significantly suppressed in saliva and elevated in GCF. The T-cell-derived cytokine IL-2 did not differ between patients and controls in serum and saliva, but there was a significant suppression in GCF of patients. Interestingly, TGF-β1 levels were significantly elevated in serum, saliva and GCF in patients compared to controls. Furthermore, by using cultured gingival fibroblasts stimulated with wild type and proteinase mutant strains of Porphyromonas gingivalis, we show that the suppression of CXCL8 and IL-6, and the induction of TGF-β1 is primarily mediated by the proteolytic activity of lysine-specific proteinases. These results indicate that P. gingivalis is a major contributor to the altered immune responses and the pathology of periodontitis. Furthermore, the ease of sampling and analyzing cytokine expression profiles, including TGF-β1, in saliva and GCF may serve to predict the progression of periodontitis and associated systemic inflammatory diseases.

Toll-like receptor 2 stimulation of platelets is mediated by purinergic P2X1-dependent Ca2+ mobilisation, cyclooxygenase and purinergic P2Y1 and P2Y12 receptor activation

Toll-like receptor 2 (TLR2), which recognise and respond to conserved microbial pathogen-associated molecular patterns, is expressed on the platelet surface. Furthermore, it has recently been shown that the TLR2/1 agonist Pam3CSK4 stimulates platelet activation. The aim of the present study was to clarify important signalling events in Pam3CSK4-induced platelet aggregation and secretion. Platelet interaction with Pam3CSK4 and the TLR2/6 agonist MALP-2 was studied by analysing aggregation, ATP-secretion, [Ca2+]i mobilisation and thromboxane B2 (TxB2) production. The results show that Pam3CSK4 but not MALP-2 induces [Ca2+]i increase, TxB2 production, dense granule secretion and platelet aggregation. Preincubation of platelets with MALP-2 inhibited the Pam3CSK4-induced responses. The ATP-secretion and aggregation in Pam3CSK4-stimulated platelets was impeded by the purinergic P2X1 inhibitor MRS 2159, the purinergic P2Y1 and P2Y12 antagonists MRS 2179 and cangrelor, the phospholipase C inhibitor U73122, the calcium chelator BAPT-AM and aspirin. The calcium mobilisation was lowered by MRS 2159, aspirin and U73122 whereas the TxB2 production was antagonised by MRS 2159, aspirin and BAPT-AM. When investigating the involvement of the myeloid differentiation factor-88 (MyD88) -dependent pathway, we found that platelets express MyD88 and interleukin 1 receptor-associated kinase (IRAK-1), which are proteins important in TLR signalling. However, Pam3CSK4 did not stimulate a rapid (within 10 minutes) phosphorylation of IRAK-1 in platelets. In conclusion, the results show that Pam3CSK4-induced platelet aggregation and secretion depends on a P2X1-mediated Ca2+ mobilisation, production of TxA2 and ADP receptor activation. The findings in this study further support a role for platelets in sensing bacterial components.