Neutrophil programming dynamics and its disease relevance

Neutrophils are traditionally considered as first responders to infection and provide antimicrobial host defense. However, recent advances indicate that neutrophils are also critically involved in the modulation of host immune environments by dynamically adopting distinct functional states. Functionally diverse neutrophil subsets are increasingly recognized as critical components mediating host pathophysiology. Despite its emerging significance, molecular mechanisms as well as functional relevance of dynamically programmed neutrophils remain to be better defined. The increasing complexity of neutrophil functions may require integrative studies that address programming dynamics of neutrophils and their pathophysiological relevance. This review aims to provide an update on the emerging topics of neutrophil programming dynamics as well as their functional relevance in diseases.     

Consequences of neutrophil adhesion to physiological and pathological targets

The ability of neutrophils to adhere in a coordinated and reversible manner to the endothelium and other tissular components is crucial to their chemoattractant-induced locomotion towards relevant targets. Opsonins play a major role in the killing effect of neutrophils by inducing close adherence between the neutrophil and the target, thus maximizing the effect of the reactive oxygen species released by the stimulated neutrophils. Reactive oxygen species are released together with degradative enzymes and other killing proteins associated with neutrophil degranulation. This targeted neutrophil activity kills invading microorganisms but, in a similar way, may be harmful to organs, cells and molecules that have been altered in some way or are involved in immune reactions. In some other pathological situations where body fluids contain proinflammatory agents, neutrophils may behave in a nontargeted and inappropriate manner. In such cases, adherence is often increased, thus slowing locomotion. Moreover, inflammatory agents often prime neutrophils for the oxidative burst induced by chemoattractants or other stimuli. The combined slow locomotion and hypersensitivity of primed neutrophils leads to a premature release of killing substances which may affect blood components, vascular cells, connective tissue or whole organs. Any disturbance of neutrophil adherence is thus potentially harmful and must be recognized and suitably treated.     

Distinct signalling mechanisms mediate neutrophil attraction to bacterial infection and tissue injury

The signals that guide neutrophils to sites of tissue injury or infection remain elusive. H(2)O(2) has been implicated in neutrophil sensing of tissue injury and transformed cells; however, its role in neutrophil recruitment to infection has not been explored. Here, using a pharmacological inhibitor of NADPH oxidases, diphenyleneiodonium (DPI), and genetic depletion of an epithelial-specific NADPH oxidase, we show that H(2)O(2) is not required for neutrophil detection of localized infection with the Gram-negative bacterium Pseudomonas aeruginosa. In contrast, PI3K signalling is required for neutrophil responses to both wounding and infection. In vivo imaging using a H(2)O(2) probe detects dynamic H(2)O(2) generation at wounds but not at infected tissue. Moreover, DPI no longer inhibits neutrophil wound attraction when P. aeruginosa is present in the media. Finally, DPI also fails to inhibit neutrophil recruitment to localized infection with the Gram-positive bacterium, Streptococcus iniae. Our findings demonstrate that different signals are involved in sensitizing neutrophils to pathogen versus non-pathogen induced tissue damage, providing a potential target to preferentially suppress non-specific immune damage without affecting the response to infection.     

Receptor for advanced glycation end-products (RAGE) modulates neutrophil adhesion and migration on glycoxidated extracellular matrix

AGEs (advanced glycation end-products) accumulate in collagen molecules during uraemia and diabetes, two diseases associated with high susceptibility to bacterial infection. Because neutrophils bind to collagen during their locomotion in extravascular tissue towards the infected area we investigated whether glycoxidation of collagen (AGE-collagen) alters neutrophil migration. Type I collagen extracted from rat tail tendons was used for in vitro glycoxidation (AGE-collagen). Neutrophils were obtained from peripheral blood of healthy adult volunteers and were used for the in vitro study of adhesion and migration on AGE- or control collagen. Glycoxidation of collagen increased adhesion of neutrophils to collagen surfaces. Neutrophil adhesion to AGE-collagen was inhibited by a rabbit anti-RAGE (receptor for AGEs) antibody and by PI3K (phosphoinositide 3-kinase) inhibitors. No effect was observed with ERK (extracellular-signal-regulated kinase) or p38 MAPK (mitogen-activated protein kinase) inhibitors. AGE-collagen was able to: (i) induce PI3K activation in neutrophils, and (ii) inhibit chemotaxis and chemokinesis of chemoattractant-stimulated neutrophils. Finally, we found that blocking RAGE with anti-RAGE antibodies or inhibiting PI3K with PI3K inhibitors restored fMLP (N-formylmethionyl-leucyl-phenylalanine)-induced neutrophil migration on AGE-collagen. These results show that RAGE and PI3K modulate adhesion and migration rate of neutrophils on AGE-collagen. Modulation of adhesiveness may account for the change in neutrophil migration rate on AGE-collagen. As neutrophils rely on their ability to move to perform their function as the first line of defence against bacterial invasion, glycoxidation of collagen may participate in the suppression of normal host defence in patients with diabetes and uraemia.     

Neutrophil stimulation and priming by direct contact with activated human T lymphocytes.

The concept that T lymphocytes regulate neutrophil function has an important implication in the understanding of the role of these cells in immunity against infection and in inflammatory diseases, but evidence for this concept is primarily derived from the effects of lymphokines on neutrophils. We now present evidence to show that living or paraformaldehyde-fixed mitogen-activated T lymphocytes, as well as an activated T cell line (HUT-78), induce by cell-cell contact, an oxygen-dependent respiratory burst measured by both the lucigenin-dependent chemiluminescence assay and superoxide production. Neutrophils reacted with purified human T lymphocytes which had been activated by culture in the presence of PHA and PMA for 72 h showed a marked and significant respiratory burst compared with neutrophils treated with T lymphocytes cultured in the absence of these mitogens. Similar results were observed with the paraformaldehyde-fixed T cell line (HUT-78). The ability to stimulate neutrophils required intact paraformaldehyde-fixed T cells, and neutrophil stimulation failed to occur if the T cells and neutrophils were separated by membrane filters. mAb to TNF-alpha, and TNF-beta blocked the ability of rTNF-alpha and TNF-beta to stimulate neutrophils but did not block the neutrophil response induced by activated T cells. Pretreatment of neutrophils with the activated T lymphocytes enhanced the response to the tripeptide, FMLP. It is therefore conceivable that activated T lymphocytes attracted at sites of inflammation influence neutrophil activity by direct plasma membrane interaction which clearly represents an efficient microbial defence mechanism, minimizing tissue damage during inflammation.     

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.     

Impact of neutrophils on antiviral activity of human bronchoalveolar lavage fluid

Surfactant protein D (SP-D) and neutrophils participate in the early innate immune response to influenza A virus (IAV) infection. SP-D increases neutrophil uptake of IAV and modulates neutrophil respiratory burst responses to IAV; however, neutrophil proteases have been shown to degrade SP-D, and human neutrophil peptide defensins bind to SP-D and can cause precipitation of SP-D from bronchoalveolar lavage fluid (BALF). BALF has significant antiviral activity against IAV. We first added neutrophils to BALF during incubation with IAV. Addition of neutrophils to BALF caused significantly greater clearance of IAV from culture supernatants than from BALF alone, and this effect was significantly more pronounced when neutrophils were activated during incubation with the virus. In contrast, if activated neutrophils were incubated with BALF before addition of virus, they reduced antiviral activity of BALF. This effect correlated with depletion of SP-D from BALF. Activation of neutrophils with agonists that induce primary granule release (including release of human neutrophil peptide defensins) caused SP-D depletion, but activation with PMA, which causes only secondary granule release, did not. The ability of activated neutrophils to deplete SP-D from BALF was partially, but not fully, corrected with protease inhibitors but was unaffected by inhibition of neutrophil respiratory burst responses. These results suggest that chronic neutrophilic inflammation (e.g., as in chronic smoking or cystic fibrosis) may reduce SP-D levels and predispose to IAV infection. In contrast, acute inflammation, as occurs in the early phase of IAV infection, may promote neutrophil-mediated viral clearance.     

Induction of ERK-kinase signalling triggers morphotype-specific killing of Candida albicans filaments by human neutrophils

Candida albicans is among the most important fungal pathogens in humans. Morphological plasticity has been linked to its pathogenic potential as filamentous forms are associated with tissue invasion and infection. Here we show that human neutrophils discriminate between yeasts and filaments of C. albicans . Whereas filaments induced targeted motility, resulting in the establishment of close contact between neutrophils and fungal cells, yeast forms were largely ignored during coincubation. In transwell assays, C. albicans filaments induced significantly higher migratory activity in neutrophils than yeasts. Neutrophil motility based on actin rearrangement was essential for killing of C. albicans filaments but not involved in killing of yeast forms. Using inhibitors for MAP-kinase cascades, it was shown that recognition of C. albicans filaments by neutrophils is mediated via the MEK/ERK cascade and independent of JNK or p38 activation. Inhibition of the ERK signalling pathway abolished neutrophil migration induced by C. albicans filaments and selectively impaired the ability to kill this morphotype. These data show that invasive filamentous forms of C. albicans trigger a morphotype-specific activation of neutrophils, which is strongly dependent on neutrophil motility. Therefore, human neutrophils are capable of sensing C. albicans invasion and initiating an appropriate early immune response.     

Identification of an immunomodulating metalloprotease of Pseudomonas aeruginosa (IMPa)

Phagocytosis by neutrophils is the essential step in fighting Pseudomonas infections. The first step in neutrophil recruitment to the site infection is the interaction of P-selectin (on endothelial cells) with P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils. Pseudomonas aeruginosa secretes various proteases that degrade proteins that are essential for host defence, such as elastase and alkaline protease. Here we identify PA0572 of P. aeruginosa as an inhibitor of PSGL-1 and named this secreted hypothetical protease immunomodulating metalloprotease of P. aeruginosa or IMPa. Proteolytic activity was confirmed by cleavage of recombinant and cell-surface expressed PSGL-1. Functional inhibition was demonstrated by impaired PSGL-1-mediated rolling of IMPa-treated neutrophils under flow conditions. Next to PSGL-1, IMPa targets CD43 and CD44 that are also involved in leucocyte homing. These data indicate that IMPa prevents neutrophil extravasation and thereby protects P. aeruginosa from neutrophil attack.     

Evidence that a receptor-operated event on the neutrophil mediates neutrophil accumulation in vivo. Pretreatment of 111In-neutrophils with pertussis toxin in vitro inhibits their accumulation in vivo

The role of neutrophil chemoattractant receptors in neutrophil stimulation in vitro is well established, however, the precise mechanisms underlying local neutrophil accumulation at inflammatory sites in vivo have not been defined. A fundamental question that remains open is whether chemoattractants act on the endothelial cell or the neutrophil to initiate the process of neutrophil migration in vivo. To address this question we have investigated whether neutrophil accumulation in vivo can occur if chemoattractant receptor occupancy is uncoupled from neutrophil stimulation. For this purpose we have used pertussis toxin (PT) as the pharmacologic tool. We have investigated the effect of in vitro pretreatment of rabbit neutrophils with PT on their responses in vitro and on their accumulation in vivo. Pretreatment of rabbit neutrophils with PT inhibited FMLP- and C5a-, but not PMA- induced increases in CD18 expression, neutrophil adherence, and degranulation in vitro. This pretreatment procedure with PT inhibited the accumulation of radiolabeled neutrophils in vivo in response to intradermally injected FMLP, C5a, C5a des Arg, leukotriene B4, IL-8, and zymosan in rabbit skin. Further, in contrast to the in vitro results, PT inhibited the PMA-induced 111In-neutrophil accumulation in vivo. Interestingly, pretreatment of neutrophils with PT also inhibited accumulation in response to intradermally injected IL-1, despite the reports that IL-1 lacks neutrophil chemoattractant activity in vitro. Although the experimental techniques used cannot distinguish the different stages of neutrophil migration involved, these results suggest that the accumulation of neutrophils induced by local extravascular chemoattractants in vivo depends on a pertussis toxin-sensitive receptor operated event on the neutrophil itself. Further, PMA and IL-1 may release secondary chemoattractants in vivo.     

The proline-rich tyrosine kinase Pyk2 modulates integrin-mediated neutrophil adhesion and reactive oxygen species generation

Neutrophils are first responders in infection and inflammation. They are able to roll, adhere and transmigrate through the endothelium to reach the site of infection, where they fight pathogens through secretion of granule contents, production of reactive oxygen species, extrusion of neutrophil extracellular traps, and phagocytosis. In this study we explored the role of the non-receptor focal adhesion kinase Pyk2 in neutrophil adhesion and activation. Using a specific Pyk2 pharmacological inhibitor, PF-4594755, as well as Pyk2-deficient murine neutrophils, we found that Pyk2 is activated upon integrin αMβ2-mediated neutrophil adhesion to fibrinogen. This process is triggered by Src family kinases-mediated phosphorylation and supported by Pyk2 autophosphorylation on Y402. In neutrophil adherent to fibrinogen, Pyk2 activates PI3K-dependent pathways promoting the phosphorylation of Akt and of its downstream effector GSK3. Pyk2 also dynamically regulates MAP kinases in fibrinogen-adherent neutrophils, as it stimulates p38MAPK but negatively regulates ERK1/2. Pharmacological inhibition of Pyk2 significantly prevented adhesion of human neutrophils to fibrinogen, and neutrophils from Pyk2-knockout mice showed a reduced ability to adhere compared to wildtype cells. Accordingly, neutrophil adhesion to fibrinogen was reduced upon inhibition of p38MAPK but potentiated by ERK1/2 inhibition. Neutrophil adherent to fibrinogen, but not to polylysine, were able to produce ROS upon lipopolysaccharide challenge and ROS production was completely suppressed upon inhibition of Pyk2. By contrast PMA-induced ROS production by neutrophil adherent to either fibrinogen or polylysine was independent from Pyk2. Altogether these results demonstrate that Pyk2 is an important effector in the coordinated puzzle regulating neutrophil adhesion and activation.     

Promotion of neutrophil apoptosis by TNF-alpha

We examined the ability of TNF-alpha to modulate human neutrophil apoptosis. Neutrophils cultured with TNF-alpha alone undergo a low but significant increase in the number of apoptotic cells. More interestingly, when neutrophils were pretreated with TNF-alpha for 1-2 min at 37 degrees C and then were exposed to a variety of agents such as immobilized IgG, IgG-coated erythrocytes, complement-treated erythrocytes, zymosan, PMA, zymosan-activated serum, fMLP, Escherichia coli, and GM-CSF for 3 h at 37 degrees C, a marked stimulation of apoptosis was observed. Similar results were obtained in neutrophils pretreated with TNF-alpha for 30 min, 1 h, 3 h, and 18 h. Dose-dependent studies showed that TNF-alpha enhances neutrophil apoptosis at concentrations ranging from 1 to 100 ng/ml. In contrast to the observations made in neutrophils pretreated with TNF-alpha, there was no stimulation of apoptosis when TNF-alpha was added to neutrophils previously activated by conventional agonists. Experiments performed to establish the mechanism through which TNF-alpha promotes neutrophil apoptosis showed that neither reactive oxygen intermediates nor the Fas/Fas ligand system appear to be involved. Our results suggest that TNF-alpha plays a critical role in the control of neutrophil survival by virtue of its ability to induce an apoptotic death program which could be triggered by a variety of conventional agonists.     

Increasing the recruitment of neutrophils to the site of infection dramatically attenuates Borrelia burgdorferi infectivity

Borrelia burgdorferi infection causes an initial skin lesion called erythema migrans (EM) in human Lyme disease and in models of monkey and rabbit borreliosis. EM results from the inflammatory response triggered by spirochete replication and likely develops to contain the initial infection but allows bacterial dissemination to occur. The essential lack of neutrophil involvement in EM histopathology prompted us to examine the consequence of increasing their recruitment in the inflammatory response to the Lyme disease agent. B. burgdorferi was modified genetically to constitutively express and secrete the chemokine KC, a neutrophil chemoattractant. After inoculation into the dermis of the murine host, control spirochetes induced an infiltration of macrophages, neutrophils, and basophils within 6 h; however, the recruited neutrophils and basophils were quickly substituted by eosinophils, and the inflammatory response became macrophage dominant by 16 h. Such a response failed to contain the initial infection and allowed the spirochetes to disseminate. In contrast, B. burgdorferi with KC secretion induced an intensive neutrophil infiltration at the inoculation site, and as a result, the host's ability to control the initial infection was greatly enhanced. Taken together, this study suggests that the failure of sufficient neutrophil recruitment and activation during the initial inflammatory response may allow B. burgdorferi to effectively colonize the mammalian host.     

Adaptive-Control Model for Neutrophil Orientation in the Direction of Chemical Gradients

Neutrophils have a remarkable ability to detect the direction of chemoattractant gradients and move directionally in response to bacterial infections and tissue injuries. For their role in health and disease, neutrophils have been extensively studied, and many of the molecules involved in the signaling mechanisms of gradient detection and chemotaxis have been identified. However, the cellular-scale mechanisms of gradient sensing and directional neutrophil migration have been more elusive, and existent models provide only limited insight into these processes. Here, we propose a what we believe is a novel adaptive-control model for the initiation of cell polarization in response to gradients. In this model, the neutrophils first sample the environment by extending protrusions in random directions and subsequently adapt their sensitivity depending on localized, temporal changes in stimulation levels. Our results suggest that microtubules may play a critical role in integrating all the sensing events from the cellular periphery through their redistribution inside the neutrophils, and may also be involved in modulating local signaling. An unexpected finding was that model neutrophils exhibit significant randomness in timing and directionality of activation, comparable to our experimental observations in microfluidic devices. Moreover, their responses are robust against alterations of the rate and amplitude of the signaling reactions, and for a broad range in chemoattractant concentrations and spatial gradients.     

C-reactive protein inhibits chemotactic peptide-induced p38 mitogen-activated protein kinase activity and human neutrophil movement.

Serum levels of the acute-phase reactant, C-reactive protein (CRP), increase dramatically during acute inflammatory episodes. CRP inhibits migration of neutrophils toward the chemoattractant, f-Met-Leu-Phe (fMLP) and therefore acts as an anti-inflammatory agent. Since tyrosine kinases are involved in neutrophil migration and CRP has been shown to decrease phosphorylation of some neutrophil proteins, we hypothesized that CRP inhibits neutrophil chemotaxis via inhibition of MAP kinase activity. The importance of p38 MAP kinase in neutrophil movement was determined by use of the specific p38 MAP kinase inhibitor, SB203580. CRP and SB203580 both blocked random and fMLP-directed neutrophil movement in a concentration-dependent manner. Additionally, extracellular signal-regulated MAP kinase (ERK) was not involved in fMLP-induced neutrophil movement as determined by use of the MEK-specific inhibitor, PD98059. Blockade of ERK with PD98059 did not inhibit chemotaxis nor did it alter the ability of CRP or SB203580 to inhibit fMLP-induced chemotaxis. More importantly, CRP inhibited fMLP-induced p38 MAP kinase activity in a concentration-dependent manner as measured by an in vitro kinase assay. Impressively, CRP-mediated inhibition of p38 MAP kinase activity correlated with CRP-mediated inhibition of fMLP-induced chemotaxis (r = -0.7144). These data show that signal transduction through p38 MAP kinase is necessary for neutrophil chemotaxis and that CRP intercedes through this pathway in inhibiting neutrophil movement.     

Human eosinophil, but not neutrophil, adherence to IL-1-stimulated human umbilical vascular endothelial cells is alpha 4 beta 1 (very late antigen-4) dependent.

Eosinophils, through their ability to generate an array of potent mediators, are thought to be the major effector cells in a number of conditions, including parasitic infection, asthma, and other allergic diseases. The mechanism(s) by which eosinophils, as opposed to neutrophils, accumulate at inflammatory sites is unknown. One possible mechanism would be an eosinophil-specific pathway of adhesion to vascular endothelium. In this study we have demonstrated that human eosinophils, but not neutrophils, constitutively express alpha 4 beta 1 (CD49d/CD29). Expression was not increased on low density eosinophils or normal density cells stimulated with platelet-activating factor. Eosinophils, but not neutrophils, specifically adhered to COS cells transfected with vascular adhesion molecule-1 in a alpha 4 beta 1-dependent manner. Eosinophil, but not neutrophil, adhesion to IL-1 stimulated human umbilical vascular endothelial cells was significantly inhibited by alpha 4 beta 1 mAb at both 5 h (p less than 0.05) and 20 h (p less than 0.001). Inhibition of both resting and platelet-activating factor-(10(-7) M) stimulated eosinophil adhesion was observed. We conclude that the alpha 4 beta 1/vascular adhesion molecule-1 adhesion pathway may be involved in specific eosinophil, as opposed to neutrophil, migration into sites of eosinophilic inflammation.     

Neutrophil adhesion molecules in experimental rhinovirus infection in COPD

Background

COPD exacerbations are associated with neutrophilic airway inflammation. Adhesion molecules on the surface of neutrophils may play a key role in their movement from blood to the airways. We analysed adhesion molecule expression on blood and sputum neutrophils from COPD subjects and non-obstructed smokers during experimental rhinovirus infections.

Methods

Blood and sputum were collected from 9 COPD subjects and 10 smoking and age-matched control subjects at baseline, and neutrophil expression of the adhesion molecules and activation markers measured using flow cytometry. The markers examined were CD62L and CD162 (mediating initial steps of neutrophil rolling and capture), CD11a and CD11b (required for firm neutrophil adhesion), CD31 and CD54 (involved in neutrophil transmigration through the endothelial monolayer) and CD63 and CD66b (neutrophil activation markers). Subjects were then experimentally infected with rhinovirus-16 and repeat samples collected for neutrophil analysis at post-infection time points.

Results

At baseline there were no differences in adhesion molecule expression between the COPD and non-COPD subjects. Expression of CD11a, CD31, CD62L and CD162 was reduced on sputum neutrophils compared to blood neutrophils. Following rhinovirus infection expression of CD11a expression on blood neutrophils was significantly reduced in both subject groups. CD11b, CD62L and CD162 expression was significantly reduced only in the COPD subjects. Blood neutrophil CD11b expression correlated inversely with inflammatory markers and symptom scores in COPD subjects.

Conclusion

Following rhinovirus infection neutrophils with higher surface expression of adhesion molecules are likely preferentially recruited to the lungs. CD11b may be a key molecule involved in neutrophil trafficking in COPD exacerbations.     

Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection

Type I inflammatory cytokines are essential for immunity to many microbial pathogens, including Toxoplasma gondii. Dendritic cells (DC) are key to initiating type 1 immunity, but neutrophils are also a source of chemokines and cytokines involved in Th1 response ignition. We found that T. gondii triggered neutrophil synthesis of CC chemokine ligand (CCL)3, CCL4, CCL5, and CCL20, chemokines that were strongly chemotactic for immature DC. Moreover, supernatants obtained from parasite-stimulated polymorphonuclear leukocytes induced DC IL-12(p40) and TNF-alpha production. Parasite-triggered neutrophils also released factors that induced DC CD40 and CD86 up-regulation, and this response was dependent upon parasite-triggered neutrophil TNF-alpha production. In vivo evidence that polymorphonuclear leukocytes exert an important influence on DC activation was obtained by examining splenic DC cytokine production following infection of neutrophil-depleted mice. These animals displayed severely curtailed splenic DC IL-12 and TNF-alpha production, as revealed by ex vivo flow cytometric analysis and in vitro culture assay. Our results reveal a previously unrecognized regulatory role for neutrophils in DC function during microbial infection, and suggest that cross-talk between these cell populations is an important component of the innate immune response to infection.