Bacterial N-formyl Peptides Research Articles

Abstract 22: Formyl Peptide Receptor-1 Activation Is Crucial For The Cause Of Spontaneous Hypertension In Dahl Salt Sensitive Rats

Mitochondria evolved from bacteria and use N-formylated peptides (NFPs) to synthetize protein. Bacterial and mitochondrial NFPs activate formyl peptide receptor 1 (FPR-1) and lead to vascular injury. We previously observed that Dahl Salt Sensitive rats (S) fed a low-salt (LS, 0.3% NaCl) diet presented spontaneous hypertension, vascular dysfunction, and overexpression of FPR-1 in arteries when compared to Dahl Salt Resistant (R) rats. High salt (HS, 2% NaCl) diet worsened these phenotypes in S rats. Interestingly, HS diet induced leaky gut and amoxicillin (AMO) treatment decreased BP in S-HS. Due to the dual sources of NFPs (microbiota and host mitochondria), we hypothesized that cell death-derived mitochondria and/or leaky gut-derived bacterial NFPs lead to FPR-1 activation, vascular injury and elevated BP in S rats independent of HS diet. For this, we used flow cytometry to measure cell necrosis and early and late apoptosis in kidney, bone marrow-derived macrophages and mesenteric resistance arteries (MRA) from male S and R rats (8-week old) on a LS diet. Zonulin, a biomarker for leaky gut, was measured in plasma. In another group, rats were treated with FPR-1 antagonist [Cyclosporin H (CsH), 0.3 mg/kg/day, osmotic mini-pump, 14 days], vehicle (VEH) or received water with AMO (5 mg/kg/day) for 21 days to deplete bacteria. BP was measured by telemetry and vascular function and structure were assessed in MRA. S rats presented increased kidney cell necrosis (R: 3.8±0.3 vs. S: 5.3±0.5* %). CsH decreased spontaneous elevation of BP [Diastolic: R+VEH: 77±2.7 vs. R+CsH: 81±1.2 vs. S+VEH: 126±3.0* vs. S+CsH:115±2.7 # ] and vascular hypercontractility [KCl (120mM): R+VEH: 9.4±1 vs. R+CsH: 10.2±0.4; S+VEH: 15.5±0.9* vs. S+CsH:11.7±0.8 # mN; Phenylephrine (10μM): R+VEH: 9.3±1 vs. R+CsH: 9.7±1; S+VEH: 14.5±1*vs. S+CsH: 11.4±0.6 # mN) in S-LS rats. AMO did not change vascular contraction or BP. Leaky gut was not observed in Dahl S-LS diet. In conclusion, FPR-1 can serve as a causative agent for the spontaneous elevation of BP and kidney-derived mitochondria, but not gut-derived microbiota, are the main source for NFPs.

Abstract 125: Low-Grade Chronic Infection Induces Vascular Dysfunction and Remodeling in Salt Sensitive Hypertension

According to the new AHA guidelines, over half of Americans have hypertension. Multiple factors contribute to hypertension including genetics, diet, stress, and infection. N-formyl peptides (NFPs) are a potent chemoattractant that bind formyl peptide receptors (FPR), an innate immune receptor. Interestingly, the only source of NFPs are bacteria and mitochondria, which evolved from bacteria. We have observed that FPR is expressed in arteries and its activation with bacterial (fMLPs) or mitochondrial (FMIT) NFPs induces vascular remodeling via actin polymerization. Given that increased gut permeability is present in hypertension, we questioned if bacterial NFPs derived from a leaky gut also play a role in hypertension. We hypothesized that bacterial NFPs activate FPR leading to vascular dysfunction and remodeling. We used male (6 weeks) Dahl Resistant (DR) and Sensitive (DS) rats on a low (0.3 %) or high salt (2 %) diet for 9 weeks. After 6 weeks some animals on high salt received water + antibiotic [neomycin (NEO), 0.5 g/L or amoxicillin (AMO), 50 mg/kg/day) for 3 weeks to decrease bacterial levels. Statistics: t-test *p<0.05. We observed that FPR mRNA expression was increased in aorta from DS (low-salt) when compared to DR (low-salt) (DR: 0.96 ± 0.7 vs. DS: 1.3 ± 1.7*), suggesting that FPR is activated prior to the onset of hypertension. High-salt diet increased FPR mRNA expression in aorta from DR, but did not exacerbate it in DS compared to DS (low-salt). Phenylephrine and acetylcholine concentration-response curves (1nM – 1μM) were performed in mesenteric resistance arteries using wire myography. Due to increased arterial stiffness from severe hypertension in DS (high salt), these rats showed a decrease in contraction compared to DR. DS treated with NEO or AMO ameliorated this response (tension (mN): Emax: DS 11 ± 4 vs. DS+ NEO 17 ± 2*, S + AMO 17 ± 0.9*). No changes were observed in the relaxation curve. Using pressure myograph, it was observed that NEO and AMO reverted vascular remodeling [wall thickness (μm): DS 55 ± 4 vs. DS + NEO 38 ± 9* and DS + AMO 31± 9*; cross sectional area (μm 2 ): DS 45,624 ± 1370 vs. DS + NEO 28,700 ± 8370* and DS + AMO 23,276 ± 9775*]. We concluded that low-grade chronic infection plays a role in vascular dysfunction and remodeling observed in hypertension.

Abstract 114: Increased Circulating Levels of Mitochondrial N-Formyl Peptides Leads to Vascular Dysfunction and High Blood Pressure in Spontaneously Hypertensive Rats

Aims: The most powerful signaling pathway that induces actin polymerization and movement in neutrophils is due to formyl peptide receptor (FPR) activation. FPR was originally identified by its ability to bind bacterial N-formyl peptides. Mitochondria carry hallmarks of their bacterial ancestry and one of these hallmarks is that this organelle uses an N-formyl-methionyl-tRNA as an initiator of protein synthesis. Consequently, mitochondrial N-formyl peptides (F-MITs) are recognized by FPR. Recently, we have observed that FPR is expressed in endothelial and vascular smooth muscle cells (VSMC). We hypothesized that increased plasma levels of F-MITs activates FPR and contributes to microvascular damage and hypertension. Methods: Male SHR (12-week old) were treated for 10 days with FPR antagonist cocktail: Cyclosporin H (CsH, 0.1 mg/kg/day i.p. ) and WRW4 (0.1 mg/kg/day i.p. ) or vehicle. Blood pressure (BP) was measured by telemetry. We also treated Wistar rats (12 week old) with F-MITs (formylated peptide corresponding to the NH2-terminus of mitochondria ND6; 0.02 mg/rat/day i.p. ) or non-formylated peptides (control) for 10 days. Blood was collected to measure F-MITs. Intrarenal arteries and mesenteric resistance arteries (MRA) were isolated and mounted in a wire myograph. Wistar VSMCs were treated with F-MITs (20 min, 10 μM) or control in the presence or absence of FPR antagonists to evaluate actin polymerization. T-test *p<0.05; n=5-6. Results and Conclusion: F-MITs plasma levels were increased in SHR (1.7-fold* vs. Wistar). FPR blockade decreased BP (SHR + vehicle: 151 ± 7.2 vs. SHR+CsH+WRW4: 138 ± 0.4* mmHg) and ameliorated acetylcholine-induced relaxation (Intrarenal; Emax: SHR+vehicle: 10 ± 5 vs. SHR+CsH+WRW4: 28 ± 6* %); (MRA; Emax: SHR+vehicle: 74±10 vs. SHR+CsH+WRW4: 99±0.6* %). F-MITs infusion increased BP in Wistar rats (Control: 133 ± 1.9 vs. F-MITs: 145 ± 4.7* mmHg) and decreased acetylcholine-induced relaxation (EC50; MRA: Control: -8.765 ± 0.05 vs. F-MIT: -8.348 ± 0.08*) (Emax; Intrarenal: Control: 63±4 vs. F-MIT: 46 ± 3* %). F-MITs induced VSMCs actin polymerization (1.8 fold* vs. control) and FPR blockers abolished this result. In conclusion, FMITs play a role in vascular dysfunction and hypertension via FPR activation.

Formyl Peptide Receptor Exerts a Sentinel Role and is Important for the Dynamic Plasticity of the Vasculature

The canonical view accepts that the vascular smooth muscle cells (VSMCs) cytoskeleton is not plastic and remains stationary during a contractile event. However, it is now recognized that VSMCs display a highly dynamic process that undergoes polymerization and depolymerization based upon cellular demand.One of the most powerful signaling pathways that induces actin polymerization, Ca2+ influx and movement in neutrophils is due to formyl peptide receptor‐1 (FPR) activation. These receptors were originally identified by their ability to bind bacterial N‐formyl peptides. Interestingly, mitochondria carry hallmarks of their bacterial ancestry and one of these hallmarks is that this organelle uses an N‐formyl‐methionyl‐tRNA as an initiator of protein synthesis. Consequently, both mitochondrial and bacterial N‐formyl peptides are recognized by FPR‐1 as ligands and are known to play a role in the activation of the innate immune system.We have observed that FPR‐1 is not only expressed in sentinel cells such as leukocytes, but also in endothelial cells and VSMCs. To better understand the role of FPR‐1 in the regulation of the vascular plasticity, we studied the hypothesis that F‐MITs (formylated peptide corresponding to the NH2‐terminus of mitochondria ND6) binds FPR‐1 and leads to actin polymerization in VSMCs similar to the observed in neutrophils when activated with bacterial fragments. On the other hand, FPR‐1 absence would disrupt VSMCs actin polymerization and decrease contraction. For this, VSMCs isolated from aorta [14 weeks male C57BL6 (WT) mice] were treated with F‐MIT (20 min, 10 μM) or nonformylated peptide (control) in the presence or absence of a cocktail of FPR antagonists (FPR‐1 antagonist, cyclosporine H: CsH, 1 μmol/L and FPR‐2 antagonist, WRW4, 10 μmol/L). We observed that F‐MIT induced VSMCs actin polymerization via a shift in the filamentous:globular actin equilibrium in favor of F‐actin (1.8 fold vs. control, p<0.05). Using confocal microscopy, it was observed that VSMCs from aorta or intrarenal arteries (resistance arteries) that do not express the FPR‐1 present disrupted F‐actin and cell migration as measured by scratch assay. Also, measurements of isometric tension in aorta, demonstrated that the absence of the FPR‐1 decreased both the first ‐(fast, Ca2+ influx) and second‐phase (slow, actin polymerization) (Figure 1) of KCl (120 mmol/L)‐induced contraction and the concentration response curve to phenylephrine (Emax: WT: 8.4±1.2 vs. FPR‐1 KO: 0.6±0.2 mN, p<0.05). Jasplakinolide, an actin polymerization inducer (0.1 μmol/L), but not RhoA activator (2 μg/mL, 3 hours) restored contraction. L‐type calcium channels (Cav1.2) and AHNAK (anchors Cav 1.2 to the actin) protein expression are decreased in VSMCs from FPR‐1 KO, and Cav 1.2 activator (BayK8644, 0.1 nmol/L −1 μmol/L) was able to restore contraction. This suggests a link between actin polymerization via FPR‐1 activation and Cav 1.2 integrity and function.We established a remarkable role for an innate immune receptor FPR‐1 in VSMCs contractility and motility. This discovery is fundamental for vascular immuno‐pathophysiology, given that FPR‐1 in VSMCs not only functions as an immune regulator and danger signal sensor, but also has an important role for the dynamic plasticity of the vasculature.Support or Funding InformationNational Institutes of Health (NIH:1K99GM118885‐01)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential neutrophil chemotactic response towards IL-8 and bacterial N-formyl peptides in term newborn infants

Background: A prerequisite for an effective innate immunity is the migrative ability of neutrophils to respond to inflammatory and infectious agents such as the intermediate interleukin (IL)-8 and the end-target formyl-methionyl-leucyl-phenylalanine (fMLP) chemoattractants. The aim was to study the chemotactic capacity of neutrophils from newborn infants and adults in response to IL-8 and the bacterial peptide fMLP.Methods: In the under-agarose cell migration assay, isolated leukocytes from healthy adults and from cord blood of healthy term newborn infants were studied with dose responses towards IL-8 and fMLP. The same number of leukocytes (1 × 105 cells), with the same distribution of neutrophils and monocytes, were analyzed in neonates and adults. Chemotaxis was distinguished from randomly migrating neutrophils, and the neutrophil pattern of migration, i.e. the migration distance and the number of migrating neutrophils per distance, was evaluated.Results: In comparison to adults, fewer neutrophils from newborn infants migrated towards IL-8 and for a shorter distance (P < .01, respectively). The number of neutrophils migrating to different gradients of fMLP, the distance they migrated, and the correlation between the number and the distance were the same for neonates and adults. Random migration did not differ in any instance.Conclusion: Chemotaxis of neutrophils from newborn infants was as co-ordinated as neutrophils from adults in response to fMLP, whereas the response to IL-8 was reduced. The differential response of neutrophils from neonates to intermediate and end-target chemoattractants could indicate a reduced infectious response.

Mitochondrial N-formyl peptides cause airway contraction and lung neutrophil infiltration via formyl peptide receptor activation

Respiratory failure is a common characteristic of systemic inflammatory response syndrome (SIRS) and sepsis. Trauma and severe blood loss cause the release of endogenous molecules known as damage-associated molecular patterns (DAMPs). Mitochondrial N-formyl peptides (F-MITs) are DAMPs that share similarities with bacterial N-formylated peptides, and are potent immune system activators. Recently, we observed that hemorrhagic shock-induced increases in plasma levels of F-MITs associated with lung damage, and that antagonism of formyl peptide receptors (FPR) ameliorated hemorrhagic shock-induced lung injury in rats. Corroborating these data, in the present study, it was observed that F-MITs expression is higher in plasma samples from trauma patients with SIRS or sepsis when compared to control trauma group. Therefore, to better understand the role of F-MITs in the regulation of lung and airway function, we studied the hypothesis that F-MITs lead to airway contraction and lung inflammation. We observed that F-MITs induced concentration-dependent contraction in trachea, bronchi and bronchioles. However, pre-treatment with mast cells degranulator or FPR antagonist decreased this response. Finally, intratracheal challenge with F-MITs increased neutrophil elastase expression in lung and inducible nitric oxide synthase and cell division control protein 42 expression in all airway segments. These data suggest that F-MITs could be a putative target to treat respiratory failure in trauma patients.

Mitochondrial N-formyl peptides induce cardiovascular collapse and sepsis-like syndrome.

Fifty percent of trauma patients who present sepsis-like syndrome do not have bacterial infections. This condition is known as systemic inflammatory response syndrome (SIRS). A unifying factor of SIRS and sepsis is cardiovascular collapse. Trauma and severe blood loss cause the release of endogenous molecules known as damage-associated molecular patterns. Mitochondrial N-formyl peptides (F-MIT) are damage-associated molecular patterns that share similarities with bacterial N-formylated peptides and are potent immune system activators. The goal of this study was to investigate whether F-MIT trigger SIRS, including hypotension and vascular collapse via formyl peptide receptor (FPR) activation. We evaluated cardiovascular parameters in Wistar rats treated with FPR or histamine receptor antagonists and inhibitors of the nitric oxide pathway before and after F-MIT infusion. F-MIT, but not nonformylated peptides or mitochondrial DNA, induced severe hypotension via FPR activation and nitric oxide and histamine release. Moreover, F-MIT infusion induced hyperthermia, blood clotting, and increased vascular permeability. To evaluate the role of leukocytes in F-MIT-induced hypotension, neutrophil, basophil, or mast cells were depleted. Depletion of basophils, but not neutrophils or mast cells, abolished F-MIT-induced hypotension. Rats that underwent hemorrhagic shock increased plasma levels of mitochondrial formylated proteins associated with lung damage and antagonism of FPR ameliorated hemorrhagic shock-induced lung injury. Finally, F-MIT induced vasodilatation in isolated resistance arteries via FPR activation; however, F-MIT impaired endothelium-dependent relaxation in the presence of blood. These data suggest that F-MIT may be the link among trauma, SIRS, and cardiovascular collapse.

Bacteria activate sensory neurons that modulate pain and inflammation

SummaryNociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviors. Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed secondary to immune activation. Here we demonstrate that bacteria directly activate nociceptors, and that the immune response mediated through TLR2, MyD88, T cells, B cells, and neutrophils/monocytes is not necessary for Staphylococcus aureus induced pain in mice. Mechanical and thermal hyperalgesia parallels live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons, in part via bacterial N-formylated peptides and the pore-forming toxin alpha-hemolysin through distinct mechanisms. Specific ablation of Nav1.8-lineage neurons, which include nociceptors, abrogated pain during bacterial infection, but concurrently increased local immune infiltration and lymphadenopathy of the draining lymph node. Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host-pathogen interactions.

Formylpeptide receptor-2 contributes to colonic epithelial homeostasis, inflammation, and tumorigenesis

Commensal bacteria and their products provide beneficial effects to the mammalian gut by stimulating epithelial cell turnover and enhancing wound healing, without activating overt inflammation. We hypothesized that N-formylpeptide receptors, which bind bacterial N-formylpeptides and are expressed by intestinal epithelial cells, may contribute to these processes. Here we report that formylpeptide receptor-2 (FPR2), which we show is expressed on the apical and lateral membranes of colonic crypt epithelial cells, mediates N-formylpeptide-dependent epithelial cell proliferation and renewal. Colonic epithelial cells in FPR2-deficient mice displayed defects in commensal bacterium-dependent homeostasis as shown by the absence of responses to N-formylpeptide stimulation, shortened colonic crypts, reduced acute inflammatory responses to dextran sulfate sodium (DSS) challenge, delayed mucosal restoration after injury, and increased azoxymethane-induced tumorigenesis. These results indicate that FPR2 is critical in mediating homeostasis, inflammation, and epithelial repair processes in the colon.

Mitochondrial Peptides Are Potent Immune Activators That Activate Human Neutrophils Via FPR-1

Tissue injury from mechanical trauma modulates innate immunity. The resultant systemic inflammatory response syndrome (SIRS) closely mimics clinical sepsis, and bacterial n-formyl peptides are septic mediators. Similar formyl peptides exist in mitochondria but little is known about their actions on human neutrophils (PMN). Mitochondria were isolated from rat heptocytes and disrupted. Soluble mitochondrial degradation products (MDP) were used to stimulate human PMN. Cytosolic calcium ([Ca]i) responses to MDP were assayed with and without antibody blockade of formyl peptide receptor 1 (FPR-1) or formyl peptide receptor like-1 (FPRL-1). Chemotaxis toward MDP was assessed in trans-wells. Oxidative burst to MDP was assessed using carboxycarboxy-2', 7'-dichlorodihydrofluorescein diacetate. MDP caused [Ca]i responses similar to those caused by formyl-Met-Leu-Phe. [Ca]i responses were completely blocked by anti-FPR-1 antibodies, showing activation occurred via the high-affinity, G-protein-coupled FPR-1 receptor. MDP acted on FPR-1 to give chemotactic responses similar to 10 nM formyl-Met-Leu-Phe. MDP also caused dose-dependent oxidative burst. Formylated mitochondrial proteins are potent immune activators. Acting through the FPR-1 receptor on professional phagocytes, MDP elicits [Ca]i release responses and Ca entry via G-protein-coupled pathways. MDP activates chemotaxis and respiratory burst. Our findings suggest a novel paradigm wherein one root cause of SIRS after trauma may be the release of mitochondrial fragments from mechanically damaged tissues. In this paradigm, mitochondrial debris "alarmins" alter host PMN phenotype, activating or suppressing immunity, predisposing to SIRS, sepsis or organ failure.

Formylpeptide Receptor Single Nucleotide Polymorphism 348T&gt;C and Its Relationship to Polymorphonuclear Leukocyte Chemotaxis in Aggressive Periodontitis

Aggressive periodontitis (AgP) is associated with impaired polymorphonuclear leukocyte (PMN) chemotaxis toward bacterial N-formylpeptides. Formylpeptide receptors (FPRs) play a major role in guiding PMNs to infection sites. Previous work revealed a significant association between FPR1 single nucleotide polymorphism (SNP) 348T>C and AgP in African Americans. We tested the hypothesis that 348T impairs PMN chemotaxis by decreasing FPR mRNA expression, thereby increasing susceptibility to AgP. Blood samples were obtained from African American subjects (37 AgP cases and 38 controls). Chemotaxis to N-formyl-methionine-leucine-phenylalanine by freshly isolated PMNs was assayed in a modified Boyden chamber. RNA was isolated from PMNs, and FPR1 gene expression was quantified by real-time polymerase chain reaction (PCR). To detect FPR1 5' SNPs, genomic DNA was isolated, and four fragments spanning the FPR1 5' region were PCR-amplified and sequenced. Haplotype associations between SNP 348T>C and 5' SNPs were analyzed. The homozygous 348T genotype was only found in AgP cases (P = 0.017; odds ratio, 18.9). Subjects with this genotype exhibited a significantly lower PMN chemotactic response relative to controls and to subjects with the 348C/C or 348T/C genotype (P <0.05). There were no significant differences in PMN FPR1 expression among subjects with the 348C/C, 348T/C, and 348T/T genotypes. Eleven FPR1 5' SNPs were detected, but none of the predicted haplotypes reflected associations with AgP or with 348T. Although the 348T/T genotype is relatively rare, it is associated with significantly impaired PMN chemotaxis and an increased risk for developing AgP in African Americans. These associations do not seem to be related to significant reductions in FPR1 transcripts in subjects expressing 348T.

FPR1 does not mediate neutrophil infiltration in experimental colitis

One hallmark of the inflammatory bowel diseases is the infiltration of neutrophils (PMN) into the mucosa. The mechanism behind this infiltration is unclear but bacterial N‐formyl peptides are candidates since these are potent chemoattractants and activating factors for PMN. We investigated this hypothesis by inducing colitis in mice deficient for the formyl peptide receptor, FPR1. FPR1‐/‐ and wild type (WT) mice were administered dextran sodium sulphate (DSS) through their drinking water for 5 days followed by one day of pure water (acute phase) or 4 days of water (recovery phase), or for 2 DSS cycles (chronic phase) with the second cycle commencing on day 22. During acute colitis FPR1‐/‐ mice lost up to 5% of their starting body weight whereas WT mice lost up to 20%. However, the histopathology was similar between the two groups at both the acute and recovery phase points. This included ulcers, PMN infiltration, and loss of crypt architecture. In contrast, during the chronic phase FPR1‐/‐ mice experienced weight loss of up to 20%, greater clinical illness, and 15% shorter colons than WT mice, all indications of a more severe disease. Whether this is reflected in differences in the histopathology remains to be evaluated. We conclude that FPR1 is not necessary for PMN infiltration into the acutely inflamed colon; however, it may play a role in chronic colitis. Funded by the CCFC and an IWK Health Centre Fellowship to SMF.

Hydroxyurea corrects the dysregulated L-selectin expression and increased H2O2 production of polymorphonuclear neutrophils from patients with sickle cell anemia

Impaired polymorphonuclear neutrophil (PMN) functions during sickle cell anemia (SCA) may have a pathogenic role in the onset of vasoocclusive events. We used flow cytometry to study, in whole blood, the adhesion molecule expression and respiratory burst of PMNs from children with SCA. Three different clinical groups were studied: (1) patients with no history of vasoocclusive events (n = 15); (2) patients with a history of vasoocclusive events (n = 17); and (3) patients receiving hydroxyurea therapy for severe vasoocclusive events (n = 9). Unstimulated PMNs showed decreased L selectin expression and increased H(2)O(2) production whatever the severity of the disease, reflecting PMN activation. This could contribute to endothelial activation reflected by abnormal plasma levels of soluble adhesion molecules (soluble intercellular adhesion molecule-1, sE selectin, and sL selectin). After stimulation with bacterial N-formyl peptides (N-formyl-methionyl-leucyl-phenylalanine [fMLP]), PMNs from untreated patients with a history of vasoocclusive events showed dysregulated L selectin shedding and increased H(2)O(2) production. Furthermore, in these patients, tumor necrosis factor priming followed by fMLP stimulation induced an H(2)O(2) production significantly higher than in the other patient groups and controls. These impairments could immobilize PMNs on the endothelium, thereby inducing reduced blood flow and fostering microvascular occlusion and vascular damage. In contrast, children treated with hydroxyurea showed near-normal basal and poststimulation H(2)O(2) production as well as normal L selectin shedding after stimulation but no change in plasma levels of soluble adhesion molecules. To our knowledge, this is the first report showing major qualitative changes of PMN abnormalities upon hydroxyurea treatment in SCA patients. This strongly suggests that PMNs are a primary target of this drug.

The role of phagocytes in HIV-related oxidative stress.

in response to a variety of stimuli, phagocytes release large quantities of reactive oxygen species (ROS), which are essential for bacterial killing. However, excessive ROS production not appropriately compensated by antioxidant molecules can lead to oxidative stress, which may also play an important role in pathogenesis of HIV infection. In fact, ROS participate in chronic inflammation, HIV replication and the apoptosis of cells of the immune system. we used flow cytometry to study, in whole blood, the activation and redox status of polymorphonuclear neutrophils (PMN) and monocytes at different stages of the disease. we showed that neutrophils and monocytes from HIV-infected patients spontaneously produced increased amounts of H2O2. This increased H2O2 production was associated with alterations of adhesion molecules expression at the cell surface, which also reflected basal activation of phagocytes from the HIV-infected patients. In monocytes, basal H2O2 production correlated with viral load. This increased ROS production was associated with changes in the expression of the antiapoptotic/antioxidant compounds Bcl-2 and thioredoxin along the course of the disease. This modulation could result from a dual regulation by oxidative stress and could explain at least in part why monocyte numbers remain relatively stable throughout the disease. Monocytes expressed a normal maximal capacity to produce ROS in optimal conditions of stimulation. In contrast, after ex vivo priming with TNFalpha or IL-8, neutrophils showed a decreased H2O2 production in response to bacterial N-formyl peptides. This latter impairment correlated with the decrease in CD4+ lymphocyte numbers and with IL-8 and IL-6 plasma levels. the increased basal ROS production by phagocytes could participate to the oxidative injury which has been implicated in the pathophysiology of HIV infection. In addition, the decreased priming of H2O2 production by neutrophils could contribute to the increased susceptibility of HIV-infected patients to bacterial infections.

N-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two N-formylpeptide receptor (FPR) subtypes. Molecular characterization of FPR2, a second mouse neutrophil FPR.

The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides. Here we show that a mouse gene named Fpr-rs2 encodes a second N-formylpeptide receptor subtype selective for neutrophils which we have provisionally named FPR2. The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2. The EC(50)s, approximately 5 microM for calcium flux and chemotaxis, were approximately 100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells. Consistent with this, fMLF induced two distinct concentration optima for chemotaxis of normal mouse neutrophils, but only the high concentration optimum for chemotaxis of neutrophils from FPR knockout mice. Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.

Defective priming of the phagocyte oxidative burst in a child with recurrent intracellular infections

Human phagocytes (polymorphonuclear neutrophils and monocytes) play a critical role in host defense against invading microorganisms. Recent studies reported that circulating phagocytes undergo a final maturation process, in particular in terms of oxidative burst, during extravasation and migration to local sites of inflammation. This process is known as priming. We report here on a nine-year-old boy with successive disseminated infections due to intracellular microorganisms ( Mycobacterium bovis, BCG, and Salmonella typhimurium). No T- or B-cell quantitative or qualitative defects were found. Polymorphonuclear neutrophil (PMN) migration and NADPH oxidase in PMNs and monocytes stimulated with various agents at optimal concentrations were normal, ruling out a leukocyte adhesion deficiency syndrome, a Chediak Higashi syndrome, and a chronic granulomatous disease. Nevertheless, the patient's PMNs and monocytes showed defective priming capacity, as measured by H 2O 2 production after pretreatment with LPS (5 μg/mL for 30 min), TNFα (100 units/mL for 30 min), or IL-8 (50 ng/mL for 30 min) in response to bacterial N-formyl peptides (fMLP 10 -6 M for 5 min). In these conditions, H 2O 2 production of PMNs and monocytes from the patient did not exceed that of the samples treated with fMLP or LPS alone, while the controls strongly produced H 2O 2. Moreover, monocytes from the patient showed an impaired capacity to kill S. typhimurium in vitro. Such an impairment could be related at least in part to the priming deficiency of phagocyte oxidative burst. This case suggests, for the first time, that in vivo priming processes are critical in host defence against intracellular pathogens.

Redox and activation status of monocytes from human immunodeficiency virus-infected patients: relationship with viral load.

Monocytes are precursors of tissue macrophages, which are major targets of human immunodeficiency virus type 1 (HIV-1) infection. Although few blood monocytes are infected, their resulting activation could play a key role in the pathogenesis of HIV disease by modulating their transendothelial migration and inducing the production of reactive oxygen species (ROS). ROS participate in chronic inflammation, HIV replication, and the apoptosis of immune system cells seen in HIV-infected subjects. Published data on monocyte activation are controversial, possibly because most studies have involved monocytes isolated from their blood environment by various procedures that may alter cell responses. We therefore used flow cytometry to study, in whole blood, the activation and redox status of monocytes from HIV-infected patients at different stages of the disease. We studied the expression of adhesion molecules, actin polymerization, and cellular levels of H2O2, Bcl-2, and thioredoxin. Basal H2O2 production correlated with viral load and was further enhanced by bacterial N-formyl peptides and endotoxin. The enhanced H2O2 production by monocytes from asymptomatic untreated patients with CD4(+) cell counts above 500/microliter was associated with a decrease in the levels of Bcl-2 and thioredoxin. In contrast, in patients with AIDS, Bcl-2 levels returned to normal and thioredoxin levels were higher than in healthy controls. Restoration of these antioxidant and antiapoptotic molecules might explain, at least in part, why monocyte numbers remain relatively stable throughout the disease. Alterations of adhesion molecule expression and increased actin polymerization could play a role in transendothelial migration of these activated monocytes.

Leukocyte polarization in cell migration and immune interactions.

Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. Prior to migration leukocytes undergo polarization, with the formation of a lamellipodium at the leading edge and a uropod at the trailing edge. This cell shape allows them to convert cytoskeletal forces into net cell-body displacement. Leukocyte chemoattractants, including chemokines, provide directional cues for leukocyte motility, and concomitantly induce polarization. Chemoattractant receptors, integrins and other adhesion molecules, cytoskeletal proteins and intracellular regulatory molecules change their cellular localization during cell polarization. A complex system of signal transduction molecules, including tyrosine kinases, lipid kinases, second messengers and members of the Rho family of small GTPases is thought to regulate the cytoskeletal rearrangements underlying leukocyte polarization and migration. The elucidation of the mechanisms and signals that control this complex reorganization will lead to a better understanding of critical questions in cell biology of leukocyte migration and polarity.

Polymorphonuclear neutrophils from human immunodeficiency virus- infected patients show enhanced activation, diminished fMLP-induced L- selectin shedding, and an impaired oxidative burst after cytokine priming

Impaired polymorphonuclear neutrophil (PMN) function may contribute to the onset of certain life-threatening bacterial and fungal infections in human immunodeficiency virus (HIV)-infected patients. Published data on PMN functional activity in HIV infection are controversial, possibly because most studies have involved PMNs isolated from their blood environment by means of various procedures that may differently affect surface receptor expression and thereby alter cellular responses. We therefore used flow cytometry to study the expression of adhesion molecules at the PMN surface, actin polymerization, and the oxidative burst of whole-blood polymorphonuclear neutrophils in 42 HIV-infected patients at different stages of the disease. These PMNs were activated in vivo, as demonstrated by increased expression of the adhesion molecule CD11b/CD18, reduced L-selectin antigen expression, increased actin polymerization, and increased H2O2 production. The alterations were present in asymptomatic patients with CD4+ cell counts greater than 500/microL and did not increase with the progression of the disease. Stimulation by bacterial N-formyl peptides showed dysregulation of L-selectin shedding and decreased H2O2 production after ex vivo priming with tumor necrosis factor alpha or interleukin-8 (IL-8). These latter impairments, which correlated with the decrease in CD4+ lymphocyte numbers and with IL-8 and IL-6 plasma levels, could contribute to the increased susceptibility of HIV-infected patients to bacterial infections.

Polymorphonuclear Neutrophils From Human Immunodeficiency Virus-Infected Patients Show Enhanced Activation, Diminished fMLP-Induced L-Selectin Shedding, and an Impaired Oxidative Burst After Cytokine Priming

Impaired polymorphonuclear neutrophil (PMN) function may contribute to the onset of certain life-threatening bacterial and fungal infections in human immunodeficiency virus (HIV)-infected patients. Published data on PMN functional activity in HIV infection are controversial, possibly because most studies have involved PMNs isolated from their blood environment by means of various procedures that may differently affect surface receptor expression and thereby alter cellular responses. We therefore used flow cytometry to study the expression of adhesion molecules at the PMN surface, actin polymerization, and the oxidative burst of whole-blood polymorphonuclear neutrophils in 42 HIV-infected patients at different stages of the disease. These PMNs were activated in vivo, as demonstrated by increased expression of the adhesion molecule CD11b/CD18, reduced L-selectin antigen expression, increased actin polymerization, and increased H2O2 production. The alterations were present in asymptomatic patients with CD4+ cell counts greater than 500/microL and did not increase with the progression of the disease. Stimulation by bacterial N-formyl peptides showed dysregulation of L-selectin shedding and decreased H2O2 production after ex vivo priming with tumor necrosis factor alpha or interleukin-8 (IL-8). These latter impairments, which correlated with the decrease in CD4+ lymphocyte numbers and with IL-8 and IL-6 plasma levels, could contribute to the increased susceptibility of HIV-infected patients to bacterial infections.