Adrenoreceptors are involved in the stimulation of neutrophils by exercise-induced circulating concentrations of Hsp72: cAMP as a potential "intracellular danger signal"

Recently, the terms "stress mediators" or "danger signals" have come to be used to describe endogenous molecules that can be released in stress situations and activate the innate immune system even in the absence of antigenic stimuli. There is evidence suggesting that extracellular heat shock proteins of 72 kDa (eHsp72), together with noradrenaline (NA), are candidates as danger signals during exercise-induced stress, interacting in the activation of neutrophils. Previous studies have shown that the post-exercise circulating concentration of eHsp72 activates the phagocytic process of neutrophils with the participation of toll-like receptor 2, but that other receptors must also be involved. The present investigation evaluates the role of adrenoreceptors in the activation of the chemotaxis, phagocytosis, and fungicidal capacity of neutrophils by the post-exercise circulating concentration of eHsp72. The results showed that intact α- and β-adrenoreceptors are necessary for the stimulation of all stages of the phagocytic process by eHsp72. Also, eHsp72 increased the intracellular levels of cAMP, suggesting that it is an "intracellular danger signal" during stress-induced activation of neutrophils mediated by extracellular heat shock proteins. These results can contribute to better understanding the mechanisms involved in the regulation of the innate immune response mediated by "danger signals" during exercise, and probably during other stress situations.     

Human resting extracellular heat shock protein 72 concentration decreases during the initial adaptation to exercise in a hot, humid environment

Heat shock protein (Hsp) 72 is a cytosolic protein that also is present in the circulation. Extracellular Hsp72 (eHsp72) is inducible by exercise and is suggested to act as a danger signal to the immune system. The adaptive response of eHsp72 to repeated exercise-heat exposures in humans remains to be determined. An intracellular animal study found a reduced Hsp72 response, with no change in resting levels, during heat stress after a single day of passive heat acclimation. The current study therefore tested whether adaptations in human eHsp72 levels would similarly occur 24 hours after a single exercise-heat exposure. Seven males completed cycle exercise (42.5% V(O2peak) for 2 hours) in a hot, humid environment (38 degrees C, 60% relative humidity) on each of 2 consecutive days. Blood samples were obtained from an antecubital vein before exercise and 0 hours and 22 hours postexercise for the analysis of eHsp72. Exercise-heat stress resulted in enhanced eHsp72, with a similar absolute increase found on both days (day 1: 1.26 ng/mL [0.80 ng/mL]; day 2: 1.29 ng/mL [1.60 ng/mL]). Resting eHsp72 decreased from rest on day 1 to day 2's 22-hour postexercise sample (P < 0.05). It is suggested that the reduction in resting eHsp72 after 2 consecutive exercise-heat exposures is possibly due to an enhanced removal from the circulation, for either immunoregulatory functions, or for improved cellular stress tolerance in this initial, most stressful period of acclimation.     

Modulation of human neutrophil function by C-reactive protein

Investigation of the effect of C-reactive protein (CRP), an acute-phase reactant, on the functional capacities of human neutrophils was carried out as the basis for elucidating the biological function of C-reactive protein. An initial stimulation at low concentrations, followed by inhibition of superoxide production, and secretion of vitamin-B12-binding protein in the presence of two stimulants, phorbol myristate acetate and concanavalin A, and of neutrophil chemotaxis with increasing concentration of CRP was observed. Correlation between modulation of cell function, at least at relatively high CRP concentrations (greater than 50 micrograms/ml) and an increase in the intracellular level of cAMP is suggested. CRP was also found to enhance neutrophil phagocytosis of particles not containing phosphorylcholine, the native CRP ligand. The proposed role of CRP in neutrophil function is one of regulation and as a negative feedback for potential cytotoxic neutrophil functions.     

The in vitro effects of histamine and metiamide on neutrophil motility and their relationship to intracellular cyclic nucleotide levels.

Histamine at concentrations of 1 x 10(-5) M to 5 x 10(-5) M consistently increased neutrophil movement as measured in Boyden chambers. This effect was entirely caused by stimulation of chemokinesis (stimulated random migration) and true chemotaxis was inhibited by these concentrations. This inhibition of chemotaxis could be abolished by pretreatment with metiamide, an H-2 receptor antagonist, and levamisole, but not by diphenylhydramine, an H-1 receptor antagonist. Metiamide at similar concentrations produced a mild stimulation of chemokinesis but has no effect on true chemotaxis. The histamine effects on neutrophil motility were associated with increased levels of intracellular cAMP wehreas cAMP levels were unaffected. Agents known to elevate intracellular cAMP levels produced effects on neutrophil motility similar to those of histamine. It is suggested that histamine exerts a 2-fold effect on neutrophil motility mediated via an H-2 receptor site and associated with elevated levels of cAMP.     

Maggot excretions/secretions inhibit multiple neutrophil pro-inflammatory responses

There is renewed interest in the use of maggots (Lucilia sericata) to aid in healing of chronic wounds. In such wounds neutrophils precipitate tissue damage rather than contribute to healing. As the molecules responsible for the beneficial actions of maggots are contained in their excretions/secretions (ES), we assessed the effects of ES on functional activities of human neutrophils. ES dose-dependently inhibited elastase release and H(2)O(2) production by fMLP-activated neutrophils; maximal inhibition was seen with 5-50 microg of ES/ml. In contrast, ES did not affect phagocytosis and intracellular killing of Candida albicans by neutrophils. Furthermore, 0.5 microg of ES/ml already inhibited neutrophil migration towards fMLP. ES dose-dependently reduced the fMLP-stimulated expression of CD11b/CD18 by neutrophils, suggesting that ES modulate neutrophil adhesion to endothelial cells. ES did not affect the fMLP-induced rise in [Ca(2+)](i) in neutrophils, indicating that ES act down-stream of phospholipase C-mediated activation of protein kinase C. In agreement, ES inhibited PMA-activated neutrophil functional activities. ES induced a rise in intracellular cAMP concentration in neutrophils and pharmacological activators of cAMP-dependent mechanisms mimicked their inhibitory effects on neutrophils. The beneficial effects of maggots on chronic wounds may be explained in part by inhibition of multiple pro-inflammatory responses of activated neutrophils by ES.     

Stimulation of human neutrophil phagocytosis of Candida albicans by a beta-glucan synthase inhibitor, cilofungin.

The effect of a lipopeptide antifungal agent, cilofungin, on serum opsonization and phagocytosis of Candida albicans yeast phase cells in human neutrophil monolayer assays was investigated. Simultaneous addition of fungicidal concentrations of cilofungin did not enhance or inhibit phagocytosis of C. albicans. Pretreatment of Candida blastospores with cilofungin in the absence of serum complement for 1 h did not affect phagocytosis. However, pretreatment of blastospores with cilofungin and complement promoted a significant increase in ingestion. Pretreatment of neutrophils with cilofungin in serum-free media did not affect neutrophil viability. In contrast, pre-exposure of neutrophils to cilofungin in the presence of complement inhibited ingestion of blastospores.     

Extracellular heat shock protein 72 is a marker of the stress protein response in acute lung injury

Previous studies have shown that heat shock protein 72 (Hsp72) is found in the extracellular space (eHsp72) and that eHsp72 has potent immunomodulatory effects. However, whether eHsp72 is present in the distal air spaces and whether eHsp72 could modulate removal of alveolar edema is unknown. The first objective was to determine whether Hsp72 is released within air spaces and whether Hsp72 levels in pulmonary edema fluid would correlate with the capacity of the alveolar epithelium to remove alveolar edema fluid in patients with ALI/ARDS. Patients with hydrostatic edema served as controls. The second objective was to determine whether activation of the stress protein response (SPR) caused the release of Hsp72 into the extracellular space in vivo and in vitro and to determine whether SPR activation and/or eHsp72 itself would prevent the IL-1beta-mediated inhibition of the vectorial fluid transport across alveolar type II cells. We found that eHsp72 was present in plasma and pulmonary edema fluid of ALI patients and that eHsp72 was significantly higher in pulmonary edema fluid from patients with preserved alveolar epithelial fluid clearance. Furthermore, SPR activation in vivo in mice and in vitro in lung endothelial, epithelial, and macrophage cells caused intracellular expression and extracellular release of Hsp72. Finally, SPR activation, but not eHsp72 itself, prevented the decrease in alveolar epithelial ion transport induced by exposure to IL-1beta. Thus SPR may protect the alveolar epithelium against oxidative stress associated with experimental ALI, and eHsp72 may serve as a marker of SPR activation in the distal air spaces of patients with ALI.     

Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing

An inability of neutrophils to eliminate invading microorganisms is frequently associated with severe infection and may contribute to the high mortality rates associated with sepsis. In the present studies, we examined whether metformin and other 5′ adenosine monophosphate-activated protein kinase (AMPK) activators affect neutrophil motility, phagocytosis and bacterial killing. We found that activation of AMPK enhanced neutrophil chemotaxis in vitro and in vivo, and also counteracted the inhibition of chemotaxis induced by exposure of neutrophils to lipopolysaccharide (LPS). In contrast, small interfering RNA (siRNA)-mediated knockdown of AMPKα1 or blockade of AMPK activation through treatment of neutrophils with the AMPK inhibitor compound C diminished neutrophil chemotaxis. In addition to their effects on chemotaxis, treatment of neutrophils with metformin or aminoimidazole carboxamide ribonucleotide (AICAR) improved phagocytosis and bacterial killing, including more efficient eradication of bacteria in a mouse model of peritonitis-induced sepsis. Immunocytochemistry showed that, in contrast to LPS, metformin or AICAR induced robust actin polymerization and distinct formation of neutrophil leading edges. Although LPS diminished AMPK phosphorylation, metformin or AICAR was able to partially decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on downstream signaling events, particularly LPS-induced IκBα degradation. The IκB kinase (IKK) inhibitor PS-1145 diminished IκBα degradation and also prevented LPS-induced inhibition of chemotaxis. These results suggest that AMPK activation with clinically approved agents, such as metformin, may facilitate bacterial eradication in sepsis and other inflammatory conditions associated with inhibition of neutrophil activation and chemotaxis.     

Exposure to perflubron is associated with decreased Syk phosphorylation in human neutrophils.

Liquid ventilation with perflubron is associated with reduced neutrophil recruitment into the lung during acute injury. Perflubron also reduces chemotactic responses, the respiratory burst, and cytokine production in neutrophils and in alveolar macrophages in vitro. In the current studies, the effect of perflubron on neutrophil chemotaxis to formyl-Met-Leu-Phe (fMLP) and phagocytosis of opsonized sheep erythrocytes (EA) correlated with decreased phosphorylation of Syk, an important intracellular second messenger in pathways regulating neutrophil functional responses. Brief (5 min) exposure of neutrophils to perflubron resulted in a dose-dependent reduction in chemotaxis to fMLP and reduced phagocytosis of EA but no apparent morphological changes as seen by electron microscopy. Concurrently, there was a reduction in both total cytosolic tyrosine phosphorylation and Syk phosphorylation. Binding studies indicated that this effect was neither a result of impaired ligand-receptor affinity nor a change in the number of fMLP receptors available on the neutrophil surface. These results suggest that perflubron nonspecifically affects cellular activation as measured by tyrosine phosphorylation perhaps by interfering with transmembrane signal transduction.     

Plasma Hsp72 (HSPA1A) and Hsp27 (HSPB1) expression under heat stress: influence of exercise intensity

Extracellular heat-shock protein 72 (eHsp72) expression during exercise-heat stress is suggested to increase with the level of hyperthermia attained, independent of the rate of heat storage. This study examined the influence of exercise at various intensities to elucidate this relationship, and investigated the association between eHsp72 and eHsp27. Sixteen male subjects cycled to exhaustion at 60% and 75% of maximal oxygen uptake in hot conditions (40°C, 50% RH). Core temperature, heart rate, oxidative stress, and blood lactate and glucose levels were measured to determine the predictor variables associated with eHsp expression. At exhaustion, heart rate exceeded 96% of maximum in both conditions. Core temperature reached 39.7°C in the 60% trial (58.9 min) and 39.0°C in the 75% trial (27.2 min) (P < 0.001). The rate of rise in core temperature was 2.1°C h⁻¹ greater in the 75% trial than in the 60% trial (P < 0.001). A significant increase and correlation was observed between eHsp72 and eHsp27 concentrations at exhaustion (P < 0.005). eHsp72 was highly correlated with the core temperature attained (60% trial) and the rate of increase in core temperature (75% trial; P < 0.05). However, no common predictor variable was associated with the expression of both eHsps. The similarity in expression of eHsp72 and eHsp27 during moderate- and high-intensity exercise may relate to the duration (i.e., core temperature attained) and intensity (i.e., rate of increase in core temperature) of exercise. Thus, the immuno-inflammatory release of eHsp72 and eHsp27 in response to exercise in the heat may be duration and intensity dependent.     

Stress-induced extracellular Hsp72 is a functionally significant danger signal to the immune system

Extracellular heat-shock proteins (eHsp) such as those belonging to the 70-kDa family of Hsp (eg, Hsp72) have been hypothesized to act as a "danger signal" to immune cells, promote immune responses, and improve host defense. The current study tested this hypothesis. Adult male F344 rats were exposed to an acute laboratory stressor (100, 5-second, 1.6-mA inescapable tail shocks) and challenged with Escherichia coli. The number of colony-forming units (CFU) of bacteria at the site of injection, the levels of eHsp72, the immune response to eHsp72 and E. coli-derived lipopolysaccharide (LPS), and the amount of time required to recover from in vivo bacterial challenge were measured. CFUs were reduced 2, 4, and 6 hours after injection of E. coli in rats exposed to stress. Rats exposed to stress had elevated eHsp72 that was elevated rapidly (25 minutes) and remained elevated in the circulation and at the inflammatory site (2 hours after stressor termination). Both stressor exposure and eHsp72 administration in the absence of stress resulted in a facilitated pattern of recovery after bacterial inflammation induced by subcutaneous E. coli injection. Rats exposed to acute restraint (100 minutes) did not demonstrate elevated circulating eHsp72 or a facilitated pattern of recovery after bacterial challenge. In vitro stimulation of rat splenocytes and macrophages with eHsp72 elevated nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, and this effect was specific to eHsp72 because it was not diminished by polymyxin B and was reduced by earlier heat-denature treatment. Stimulation of cells with eHsp72 combined with LPS resulted in a greater NO and cytokine response than that observed after stimulation with eHsp72 or LPS alone. In vivo, at the inflammatory site, the bacterial-induced NO response was potentiated by stress, and NO inhibition (L-NIO) reduced the stress-induced facilitation but had no effect on the control kinetics of bacterial inflammation recovery. Thus, these results lend support to the hypothesis that intense stressor exposure increases eHsp72, which acts as a danger signal to potentiate the NO response to bacterial challenge and facilitate recovery from bacterial inflammation.     

Effects of aminopeptidase inhibitors actinonin and amastatin on chemotactic and phagocytic responses of human neutrophils

Actinonin and amastatin are low-molecular-weight inhibitors of aminopeptidases associated with cell surfaces. The purpose of this study was to determine their effects on human neutrophil functions such as chemotaxis and phagocytosis. Both actinonin and amastatin enhanced chemotaxis to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. On the other hand, the effects of both agents on neutrophil phagocytosis were varied. Bacterial attachment to neutrophils was slightly affected by these agents. However, actinonin enhanced the internalization of bacteria by neutrophils. Neutrophil leucine aminopeptidase activity was also determined and was found to be weakly inhibited by these agents.     

Extracellular Hsp72 concentration relates to a minimum endogenous criteria during acute exercise-heat exposure

Extracellular heat shock protein 72 (eHsp72) concentration increases during exercise-heat stress when conditions elicit physiological strain. Differences in severity of environmental and exercise stimuli have elicited varied response to stress. The present study aimed to quantify the extent of increased eHsp72 with increased exogenous heat stress, and determine related endogenous markers of strain in an exercise-heat model. Ten males cycled for 90 min at 50 % in three conditions (TEMP, 20 °C/63 % RH; HOT, 30.2 °C/51%RH; VHOT, 40.0 °C/37%RH). Plasma was analysed for eHsp72 pre, immediately post and 24-h post each trial utilising a commercially available ELISA. Increased eHsp72 concentration was observed post VHOT trial (+172.4 %) (p < 0.05), but not TEMP (−1.9 %) or HOT (+25.7 %) conditions. eHsp72 returned to baseline values within 24 h in all conditions. Changes were observed in rectal temperature (T_rec), rate of T_rec increase, area under the curve for T_rec of 38.5 and 39.0 °C, duration T_rec ≥ 38.5 and ≥39.0 °C, and change in muscle temperature, between VHOT, and TEMP and HOT, but not between TEMP and HOT. Each condition also elicited significantly increasing physiological strain, described by sweat rate, heart rate, physiological strain index, rating of perceived exertion and thermal sensation. Stepwise multiple regression reported rate of T_rec increase and change in T_rec to be predictors of increased eHsp72 concentration. Data suggests eHsp72 concentration increases once systemic temperature and sympathetic activity exceeds a minimum endogenous criteria elicited during VHOT conditions and is likely to be modulated by large, rapid changes in core temperature.     

mTORC2 regulates neutrophil chemotaxis in a cAMP- and RhoA-dependent fashion

We studied the role of the target of rapamycin complex 2 (mTORC2) during neutrophil chemotaxis, a process that is mediated through the polarization of actin and myosin filament networks. We show that inhibition of mTORC2 activity, achieved via knock down (KD) of Rictor, severely inhibits neutrophil polarization and directed migration induced by chemoattractants, independently of Akt. Rictor KD also abolishes the ability of chemoattractants to induce cAMP production, a process mediated through the activation of the adenylyl cyclase 9 (AC9). Cells with either reduced or higher AC9 levels also exhibit specific and severe tail retraction defects that are mediated through RhoA. We further show that cAMP is excluded from extending pseudopods and remains restricted to the cell body of migrating neutrophils. We propose that the mTORC2-dependent regulation of MyoII occurs through a cAMP/RhoA-signaling axis, independently of actin reorganization during neutrophil chemotaxis.     

The endogenous opioid spinorphin blocks fMet-Leu-Phe-induced neutrophil chemotaxis by acting as a specific antagonist at the N-formylpeptide receptor subtype FPR.

Spinorphin is an endogenous heptapeptide (leucylvalylvalyltyrosylprolyltryptophylthreonine), first isolated from bovine spinal cord, whose sequence matches a conserved region of beta-hemoglobin. Also referred to as LVV-hemorphin-4 and a member of the nonclassical opioid hemorphin family, spinorphin inhibits enkephalin-degrading enzymes and is analgesic. Recently, spinorphin was reported to block neutrophil activation induced by the chemotactic N-formylpeptide N-formylmethionylleucylphenylalanine (fMLF), suggesting a potential role as an endogenous negative regulator of inflammation. Here we use both gain- and loss-of-function genetic tests to identify the specific mechanism of spinorphin action on neutrophils. Spinorphin induced calcium flux in normal mouse neutrophils, but was inactive in neutrophils from mice genetically deficient in the fMLF receptor subtype FPR (N-formylpeptide receptor). Consistent with this, spinorphin induced calcium flux in human embryonic kidney 293 cells transfected with mouse FPR, but had no effect on cells expressing the closely related fMLF receptor subtype FPR2. Despite acting as a calcium-mobilizing agonist at FPR, spinorphin was a weak chemotactic agonist and effectively blocked neutrophil chemotaxis induced by fMLF at concentrations selective for FPR. Spinorphin did not affect mouse neutrophil chemotaxis induced by concentrations of fMLF that selectively activate FPR2. Thus, spinorphin blocks fMLF-induced neutrophil chemotaxis by acting as a specific antagonist at the fMLF receptor subtype FPR.     

Mycobacterial cord factor enhances migration of neutrophil‐like HL‐60 cells by prolonging AKT phosphorylation

Trehalose 6,6′‐dimycolate (TDM), or cord factor, is a crucial stimulus of immune responses during Mycobacterium tuberculosis infection. Although TDM has immuno‐stimulatory properties, including adjuvant activity and the ability to induce granuloma formation, the mechanisms underlying these remain unknown. We hypothesized that TDM stimulates transendothelial migration of neutrophils, which are the first immune cells to infiltrate the tissue upon infection. In this study, it was shown that TDM enhances N‐formylmethionyl‐leucyl‐phenylalanine (fMLP)‐induced chemotaxis and transendothelial movement by prolonging AKT phosphorylation in human neutrophils. TDM induced expression of macrophage‐inducible C‐type lectin, a receptor for TDM, and induced secretion of pro‐inflammatory cytokines and chemokines in differentiated HL‐60 cells. In 2‐ and 3‐D neutrophil migration assays, TDM‐stimulated neutrophils showed increased fMLP‐induced chemotaxis and transendothelial migration. Interestingly, following fMLP stimulation of TDM‐activated neutrophils, AKT, a crucial kinase for neutrophil polarization and chemotaxis, showed prolonged phosphorylation at serine 473. Taken together, these data suggest that TDM modulates transendothelial migration of neutrophils upon mycobacterial infection through prolonged AKT phosphorylation. AKT may therefore be a promising therapeutic target for enhancing immune responses to mycobacterial infection.

     

Elevation of body temperature is an essential factor for exercise-increased extracellular heat shock protein 72 level in rat plasma

This study examined whether the exercise-increased extracellular heat shock protein 72 (eHsp72) levels in rats was associated with body temperature elevation during exercise. In all, 26 female Sprague-Dawley rats (3 mo old) were assigned randomly to control (CON; n = 8), exercise under warm temperature (WEx; n = 9), or exercise under cold temperature (CEx; n = 9). The WEx and CEx were trained at 25 degrees C or 4 degrees C, respectively, for nine days using a treadmill. Before and immediately after the final exercise bout, the colonic temperatures were measured as an index of body temperature. The animals were subsequently anesthetized, and blood samples were collected and centrifuged. Plasma samples were obtained to assess their eHsp72 levels. Only the colonic temperature in WEx was increased significantly (P < 0.05) by exercise. The eHsp72 level in WEx was significantly higher (P < 0.05) than that of either the CON or CEx. However, no significant difference was found between CON and CEx. Regression analyses revealed that the eHsp72 level increased as a function of the body temperature. In another experiment, the eHsp72 level of animals with body temperature that was passively elevated through similar kinetics to those of the exercise was studied. Results of this experiment showed that mere body temperature elevation was insufficient to induce eHsp72 responses. Collectively, our results suggest that body temperature elevation during exercise is important for induction of exercise-increased eHsp72. In addition, the possible role of body temperature elevation is displayed when the exercise stressor is combined with it.     

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.     

Damage-associated molecular pattern S100A9 increases bactericidal activity of human neutrophils by enhancing phagocytosis

The damage-associated molecular-pattern S100A9 is found at inflammatory sites in infections and various autoimmune diseases. It is released at very high concentrations in the extracellular milieu by activated neutrophils and monocytes in response to various agents. This proinflammatory protein is found in infected mucosae and tissue abscesses where it acts notably as a potent neutrophil activator. In this study, we examined the role of S100A9 in the control of infections. S100A9 was found to increase human neutrophil bactericidal activity toward Escherichia coli. Although S100A9 induced the accumulation of reactive oxygen species over time through the activation of NADPH oxidase, its antimicrobial activity was mediated mainly by enhancing the efficiency of neutrophil phagocytosis. Interestingly, S100A9 did not act by increasing cell surface expression of CD16, CD32, or CD64 in neutrophils, indicating that its biological effect in FcR-mediated phagocytosis is independent of upregulation of FcγR levels. However, S100A9-induced phagocytic activity required the phosphorylation of Erk1/2, Akt, and Syk. Taken together, our results demonstrate that S100A9 stimulates neutrophil microbicidal activity by promoting phagocytosis.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.